Mammalian alpha-kinase proteins, nucleic acids and diagnostic and therapeutic uses thereof

ABSTRACT

The present invention provides novel mammalian alpha-kinase proteins: melanoma alpha-kinase (MK), heart alpha-kinase (HK), kidney alpha-kinase (KK), skeletal muscle alpha-kinase (SK), and lymphocyte alpha-kinase (LK). In particular, a novel kinase type is herein provided, characterized by the presence of an alpha-kinase catalytic domain and an ion channel domain. Isolated nucleic acids of the alpha-kinases MK, HK, KK, SK and LK are provided. Methods for making the novel alpha-kinases, cells that express the alpha-kinases and methods for treating an animal in need of either increased or decreased activity of the alpha-kinases are provided.

RELATED APPLICATIONS

The present application is a continuation-in-part of copendingapplication Ser. No. 09/632,131 filed Aug. 3, 2000, of which the instantapplication claims the benefit of the filing date pursuant to 35 U.S.C.§ 120, and which is incorporated herein by reference in its entirety.The present application claims priority pursuant to 35 U.S.C. § 119(e)to provisional application Ser. No. 60/188,957 filed Mar. 13, 2000,which is incorporated herein by reference in its entirety.

GOVERNMENTAL SUPPORT

The research leading to the present invention was supported, at least inpart, by a grant from the National Institutes of Health, Grant No.CA81102 and Grant No. GM57300. Accordingly, the Government may havecertain rights in the invention.

FIELD OF THE INVENTION

This invention relates generally to the identification of a newsuperfamily of eukaryotic protein alpha kinases, and particularly tomembers of a subfamily selected from the group of melanoma alpha kinase,kidney alpha kinase, heart alpha kinase, skeletal muscle alpha kinaseand lymphocyte alpha kinase. The invention further relates to the use ofthe alpha kinases in assays to screen for specific modulators thereof.Isolated nucleic acids encoding the alpha kinases—melanoma alpha kinase,kidney alpha kinase, heart alpha kinase, skeletal muscle alpha kinaseand lymphocyte alpha kinase—are provided herein.

BACKGROUND OF THE INVENTION

Protein phosphorylation plays a critical role in many cellular processes(Krebs (1994) Trends Biochem. Sci. 19:439; Hanks and Hunter, (1996)FASEB J 9:576–596; Hardie and Hanks, (1995) The Protein Kinase FactsBook (Academic, London)). There are two well-characterized superfamiliesof protein kinases, with most of the protein kinases belonging to theserine/threonine/tyrosine kinase superfamily (Hanks and Hunter, (1996);Hardie and Hanks, (1995)). The characterization of several hundredmembers of this superfamily revealed that they all share a similarstructural organization of their catalytic domains which consist oftwelve conserved subdomains (Hanks and Hunter, (1996); Hardie and Hanks,(1995)). The other superfamily is referred to as the histidine kinasesuperfamily and is involved in the prokaryotic two-component signaltransduction system, acting as sensor components (Stock et al., (1989)Microbiol. Rev. 53:450–490; Parkinson and Kofoid, (1992) Annu. Rev.Genet. 26:71–112; Swanson, et al., (1994) Trends Biochem. Sci.19:485–490). Recently, eukaryotic members of this superfamily have alsobeen described (Chang et al., (1993) Science 263:539–544; Ota andVarshavsky, (1993) Science 262:566–569; Maeda et al., (1994) Nature369:242–245). Mitochondrial protein kinases have also recently beendescribed that show structural homology to the histidine kinases, butphosphorylate their substrates on serine (Popov et al., (1992) J. Biol.Chem. 267:13127–13130; Popov et al., (1993) J. Biol. Chem.268:26602–22606). Finally, several new protein kinases have beenreported that show a lack of homology with either of the kinasesuperfamilies (Maru and Witte, (1991) Cell 67:459–468; Beeler et al.,(1994) Mol. Cell, Biol. 14:982–988; Dikstein et al., (1996) Cell84:781–790; Futey et al., (1995) J. Biol. Chem. 270:523–529; Eichengeret al., (1996) EMBO J. 15:5547–5556). However, these protein kinases areviewed as an exception to the general rule as they have yet to be fullycharacterized.

The cloning and sequencing of the extensively characterized eukaryoticelongation factor-2 kinase (eEF-2 kinase) from a variety of eukaryoticorganisms has revealed the existence of a novel class of protein kinases(Ryazanov et al., (1997) Proc. Natl. Acad. Sci., USA 94:4884–4889).eEF-2 kinase, previously known as Ca²⁺/calmodulin-dependent proteinkinase III, is highly specific for phosphorylation of elongationfactor-2 (eEF-2), an abundant cytoplasmic protein that catalyzes themovement of the ribosome along mRNA during translation in eukaryoticcells (reviewed in Ryazanov and Spirin, (1993) In TranslationalRegulation of Gene Expression (Plenum, New York) Vol. 2, pp. 433–455;Nairn and Palfrey, (1996) In Translational Control (CSHL Press, NewYork) pp. 295–318). All mammalian tissues, and various invertebrateorganisms, exhibit eEF-2 kinase activity (Abdelmajid et al., (1993) Int.J Dev. Biol. 37:279–290). eEF-2 kinase catalyzes the phosphorylation ofeEF-2 at two highly conserved threonine residues located within aGTP-binding domain (Ryazanov and Spirin, (1993) In TranslationalRegulation of Gene Expression (Plenum, New York) Vol. 2, pp. 433–455;Nairn and Palfrey, (1996) In Translational Control (CSHL Press, NewYork) pp. 295–318). When eEF-2 is phosphorylated, it becomes inactivewith respect to protein synthesis (Ryazanov et al., (1988) Nature334:170–173). Since eEF-2 phosphorylation is dependent on Ca²⁺ andcalmodulin, eEF-2 kinase plays a pivotal role in modulating the proteinsynthesis rate in response to changes in intracellular calciumconcentration. Phosphorylation of eEF-2 has also been linked to theregulation of cell cycle progression. For example, transientphosphorylation of eEF-2 occurs during the mitogenic stimulation ofquiescent cells (Palfrey et al., (1987) J. Biol. Chem. 262:9785–9792)and during mitosis (Celis et al., (1990) Proc. Natl. Acad. Sci., USA87:4231–4235). In addition, changes in the level of eEF-2 kinaseactivity is associated with a host of cellular processes such ascellular differentiation (End et al., (1982) J. Biol. Chem.257:9223–9225; Koizumi et al., (1989) FEBS Lett. 253:55–58; Brady etal., (1990) J. Neurochem. 54:1034–1039), oogenesis (Severinov et al.,(1990) New Biol. 2: 887–893), and malignant transformation (Bagaglio etal., (1993) Cancer Res. 53:2260–2264).

The sequence of eEF-2 kinase appears to have no homology to either theCa²⁺/calmodulin-dependent protein kinases or to any members of the knownprotein kinase superfamilies (Ryazanov et al., (1997) Proc. Natl. Acad.Sci., USA 94:4884–4889). However, the recently described myosin heavychain kinase A (MHCK A) from Dictyostelium (Futey et al., (1995) J.Biol. Chem. 270:523–529) shows a great deal of homology with eEF-2kinase. These two kinases define a novel class of protein kinases thatmay represent a new superfamily.

Evidence for MHCK and eEF-2 kinase forming the core of a new superfamilyis as follows. MHCK A from Dictyostelium, has a demonstrated role in theregulation of myosin assembly (Futey et al., (1995) J. Biol. Chem.270:523–529; Côté et al., (1997) J. Biol. Chem. 272:6846–6849). eEF-2kinase is a ubiquitous Ca²⁺/calmodulin-dependant protein kinase involvedin the regulation of protein synthesis by Ca²⁺ (Redpath et al., (1996)J. Biol. Chem 271:17547–17554; Ryazanov et al., (1997) Proc. Natl. Acad.Sci., USA 94:4884–4889). Both MHCK A and eEF-2 kinase display nohomology to any of the known protein kinases, but are strikingly similarto each other; amino acid sequences of their catalytic domains are 40%identical. Another protein kinase homologous to MHCK A and eEF-2 kinasehas recently been identified in Dictyostelium (Clancy et al., (1997) J.Biol. Chem. 272:11812–11815), and an expressed sequence tag (EST)sequence, with a high degree of similarity to the catalytic domaincommon to both MHCK A and eEF-2 kinase, has been deposited in GenBank(clone FC-AN09/accession #C22986). An amino acid sequence alignment ofthe catalytic domains of these new protein kinases is shown in FIG. 1A.These kinases have a catalytic domain of approximately 200 amino acidswhich can be subdivided into seven conserved subdomains. Subdomains V,VI, and VII have a predicted β-sheet structure and are presumablyinvolved in ATP-binding, while subdomains I through IV may be involvedin substrate binding and catalysis. These new protein kinases have nohomology to the members of the eukaryotic serine/threonine/tyrosineprotein kinase superfamily with the exception of the GXGXXG (SEQ IDNO:21) motif in subdomain VI which is present in many ATP-bindingproteins. Thus, MHCK A, eEF-2 kinase, and related protein kinases mayrepresent a new superfamily. Evolutionary analysis of these new kinases(FIG. 1B) reveals that they can be subdivided into 2 families: the eEF-2kinase family which includes eEF-2 kinases from different organisms, andthe MHCK family which includes MHCK A, MHCK B and FC-AN09. These twofamilies appear to have split more than a billion years ago.

An interesting question is why does nature employ these unusual kinasesto phosphorylate eEF-2 and myosin heavy chains? Perhaps the answer isrelated to the secondary structure of the phosphorylation sites. As wasoriginally reported by Small et al. (Small et al., (1977), Biochim.Biophys. Res. Comm. 79:341–346), phosphorylation sites are usuallylocated at predicted β-turns. Subsequent studies, including X-raycrystallographic data, demonstrated that phosphoacceptor sites insubstrates of conventional protein kinases are often located in turns orloops and usually have flexible extended conformation (Knighton et al.,(1991) Science 253:414–420; Pinna and Ruzzene (1996) Biochim. Biophys.Acta 1314:191–225). In contrast to this, the existing evidence suggeststhat the peptides around phosphorylation sites for eEF-2 kinases andMHCK A have an α-helical conformation. The two major phosphorylationsites for MHCK A are located in a region which has a coiled-coilα-helical structure (Vaillancourt et al., (1988) J. Biol. Chem.253:10082–10087). The major phosphorylation site in eEF-2, threonine 56,is located within a sequence which is homologous among all translationalelongation factors. In the crystal structure of the prokaryoticelongation factor EF-Tu, this sequence has an α-helical conformation(Polekhina et al., (1996) Structure 4:1141–1151; Abel et al., (1996)Structure 4:1153–1159). These facts suggest that eEF-2 kinase and MHCK Adiffer from conventional protein kinases in that they phosphorylateamino acids located within α-helices. Thus, in addition to the twowell-characterized superfamily of eukaryotic protein kinases, whichphosphorylate amino acids located in loops and turns, there appears tobe a third superfamily of α-helix-directed kinases.

The existence of several protein kinases which have very little or nohomology to either the serine/threonine/tyrosine kinase superfamily orthe histidine kinase superfamily, provides a new superfamily, theα-kinases. The isolation and analysis of additional members of thisfamily of kinases will further our understanding of α-kinases andprovide insight into the physiological roles of these kinases and theirapplications and uses.

The citation of references herein shall not be construed as an admissionthat such is prior art to the present invention.

SUMMARY OF THE INVENTION

In accordance with the present invention, a new superfamily of proteinkinases, novel members thereof, and corresponding methods for assayingtheir phosphorylation activity are disclosed. The protein kinases ofthis new alpha-kinase superfamily have the following characteristics: 1)No significant sequence homology to protein kinases of either theserine/threonine/tyrosine kinase or histidine kinase super families; 2)moderate to high homology (≧40%) to eEF-2 kinases from any organism;and, 3) the ability to phosphorylate an amino acid within an a-helicaldomain. In addition, a new subfamily of alpha-kinases is hereinprovided. In particular, a subfamily of alpha-kinases is provided inwhich an ion channel, particularly belonging to the TRP family of ionchannels is covalently linked to a protein kinase. The placement of akinase and channel on a single molecule is particularly interesting andsuggests a self-regulated molecule, whereby thephosphorylation/autophosphorylation of these unique alpha kinasescontrols or contributes to the open or closed state of the channel.

The present invention provides an isolated nucleic acid encodingmelanoma alpha kinase, or a fragment thereof having at least 15nucleotides. In particular, the invention provides an isolated nucleicacid encoding human melanoma alpha kinase, wherein the nucleic acid isselected from the group consisting of:

-   -   a. the DNA sequence of SEQ ID NO: 26;    -   b. DNA sequences that hybridize to the sequence of subpart (a)        under moderate stringency hybridization conditions;    -   c. DNA sequences capable of encoding the amino acid sequence        encoded by the DNA sequences of (a) or (b);    -   d. degenerate variants thereof;    -   e. alleles thereof; and    -   f. hybridizable fragments thereof.

In particular, the invention provides an isolated nucleic acid encodingmouse melanoma alpha kinase, wherein the nucleic acid is selected fromthe group consisting of:

-   -   a. the DNA sequence of SEQ ID NO: 28;    -   b. DNA sequences that hybridize to the sequence of subpart (a)        under moderate stringency hybridization conditions;    -   c. DNA sequences capable of encoding the amino acid sequence        encoded by the DNA sequences of (a) or (b);    -   d. degenerate variants thereof;    -   e. alleles thereof; and    -   f. hybridizable fragments thereof.

In particular, the invention provides an isolated nucleic acid encodingmammalian melanoma alpha kinase, wherein the nucleic acid is selectedfrom the group consisting of:

-   -   a. the DNA sequence of SEQ ID NO: 28;    -   b. the DNA sequence of SEQ ID NO: 26;    -   c. DNA sequences that hybridize to the sequence of subparts (a)        or (b) under standard hybridization conditions; and    -   d. DNA sequences capable of encoding the amino acid sequence        encoded by the DNA sequences of subparts (a), (b) or (c).

The present invention further provides an isolated nucleic acid encodingheart alpha kinase, or a fragment thereof having at least 15nucleotides. In particular, the present invention provides an isolatednucleic acid encoding human heart alpha kinase, wherein the nucleic acidis selected from the group consisting of:

-   -   a. nucleic acid comprising the DNA sequence of SEQ ID NO: 34;    -   b. DNA sequences that hybridize to the sequence of subpart (a)        under moderate stringency hybridization conditions;    -   c. DNA sequences capable of encoding the amino acid sequence        encoded by the DNA sequences of (a) or (b);    -   d. degenerate variants thereof;    -   e. alleles thereof; and    -   f. hybridizable fragments thereof.

In particular, the present invention provides an isolated nucleic acidencoding mouse heart alpha kinase, wherein the nucleic acid is selectedfrom the group consisting of:

-   -   a. nucleic acid comprising the DNA sequence of SEQ ID NO: 36;    -   b. DNA sequences that hybridize to the sequence of subpart (a)        under moderate stringency hybridization conditions;    -   c. DNA sequences capable of encoding the amino acid sequence        encoded by the DNA sequences of (a) or (b);    -   d. degenerate variants thereof;    -   e. alleles thereof; and    -   f. hybridizable fragments thereof.

In particular, the invention provides an isolated nucleic acid encodingmammalian heart alpha kinase, wherein the nucleic acid is selected fromthe group consisting of:

-   -   a. the DNA sequence of SEQ ID NO: 34;    -   b. the DNA sequence of SEQ ID NO: 36;    -   c. DNA sequences that hybridize to the sequence of subparts (a)        or (b) under standard hybridization conditions; and    -   d. DNA sequences capable of encoding the amino acid sequence        encoded by the DNA sequences of subparts (a), (b) or (c).

The present invention still further provides an isolated nucleic acidencoding kidney alpha kinase, or a fragment thereof having at least 15nucleotides. In particular, the invention includes an isolated nucleicacid encoding human kidney alpha kinase, wherein the nucleic acid isselected from the group consisting of:

-   -   a. the DNA sequence of SEQ ID NO: 30;    -   b. DNA sequences that hybridize to the sequence of subpart (a)        under moderate stringency hybridization conditions;    -   c. DNA sequences capable of encoding the amino acid sequence        encoded by the DNA sequences of (a) or (b);    -   d. degenerate variants thereof;    -   e. alleles thereof; and    -   f. hybridizable fragments thereof.

In particular, the invention includes an isolated nucleic acid encodingmouse kidney alpha kinase, wherein the nucleic acid is selected from thegroup consisting of:

-   -   a. the DNA sequence of SEQ ID NO: 32;    -   b. DNA sequences that hybridize to the sequence of subpart (a)        under moderate stringency hybridization conditions;    -   c. DNA sequences capable of encoding the amino acid sequence        encoded by the DNA sequences of (a) or (b);    -   d. degenerate variants thereof;    -   e. alleles thereof; and    -   f. hybridizable fragments thereof.

In particular, the invention provides an isolated nucleic acid encodingmammalian kidney alpha kinase, wherein the nucleic acid is selected fromthe group consisting of

-   -   a. the DNA sequence of SEQ ID NO: 30;    -   b. the DNA sequence of SEQ ID NO: 32;    -   c. DNA sequences that hybridize to the sequence of subparts (a)        or (b) under standard hybridization conditions; and    -   d. DNA sequences capable of encoding the amino acid sequence        encoded by the DNA sequences of subparts (a), (b) or (c).

The present invention also provides an isolated nucleic acid encodingskeletal muscle alpha kinase, or a fragment thereof having at least 15nucleotides. In particular, an isolated nucleic acid encoding skeletalmuscle alpha kinase is provided, wherein the nucleic acid is selectedfrom the group consisting of:

-   -   a. nucleic acid comprising the DNA sequence of SEQ ID NO: 38;    -   b. DNA sequences that hybridize to the sequence of subpart (a)        under moderate stringency hybridization conditions;    -   c. DNA sequences capable of encoding the amino acid sequence        encoded by the DNA sequences of (a) or (b);    -   d. degenerate variants thereof;    -   e. alleles thereof; and    -   f. hybridizable fragments thereof.

In particular, the invention provides an isolated nucleic acid encodingmammalian skeletal muscle alpha kinase, wherein the nucleic acid isselected from the group consisting of:

-   -   a. the DNA sequence of SEQ ID NO: 38;    -   b. DNA sequences that hybridize to the sequence of subpart (a)        under standard hybridization conditions; and    -   c. DNA sequences capable of encoding the amino acid sequence        encoded by the DNA sequences of (a) or (b).

The present invention also includes an isolated nucleic acid encodinglymphocyte alpha kinase, or a fragment thereof having at least 15nucleotides. In particular, the present invention provides an isolatednucleic acid encoding lymphocyte alpha kinase, wherein the nucleic acidis selected from the group consisting of:

-   -   a. nucleic acid comprising the DNA sequence of SEQ ID NO: 40;    -   b. DNA sequences that hybridize to the sequence of subpart (a)        under moderate stringency hybridization conditions;    -   c. DNA sequences capable of encoding the amino acid sequence        encoded by the DNA sequences of (a) or (b);    -   d. degenerate variants thereof;    -   e. alleles thereof; and    -   f. hybridizable fragments thereof.

In particular, the invention provides an isolated nucleic acid encodingmammalian lymphocyte alpha kinase, wherein the nucleic acid is selectedfrom the group consisting of:

-   -   a. the DNA sequence of SEQ ID NO: 40;    -   b. DNA sequences that hybridize to the sequence of subpart (a)        under standard hybridization conditions; and    -   c. DNA sequences capable of encoding the amino acid sequence        encoded by the DNA sequences of (a) or (b).

The invention provides an isolated nucleic acid encoding human melanomaalpha kinase, wherein the nucleic acid comprises the DNA sequence of SEQID NO: 26. The invention provides an isolated nucleic acid encodingmouse melanoma alpha kinase, wherein the nucleic acid comprises the DNAsequence of SEQ ID NO: 28.

The invention provides an isolated nucleic acid encoding human heartalpha kinase, wherein the nucleic acid comprises the DNA sequence of SEQID NO: 34. The invention provides an isolated nucleic acid encodingmouse heart alpha kinase, wherein the nucleic acid comprises the DNAsequence of SEQ ID NO: 36.

The invention provides an isolated nucleic acid encoding human kidneyalpha kinase, wherein the nucleic acid comprises the DNA sequence of SEQID NO: 30. The invention provides an isolated nucleic acid encodingmouse kidney alpha kinase, wherein the nucleic acid comprises the DNAsequence of SEQ ID NO: 32.

The invention provides an isolated nucleic acid encoding human skeletalmuscle alpha kinase, wherein the nucleic acid comprises the DNA sequenceof SEQ ID NO: 38.

The invention provides an isolated nucleic acid encoding humanlymphocyte alpha kinase, wherein the nucleic acid comprises the DNAsequence of SEQ ID NO: 40.

The present invention also relates to a recombinant DNA molecule orcloned gene, or a degenerate variant thereof, which encodes an alphakinase selected from the group of melanoma kinase, heart kinase, kidneykinase, skeletal muscle kinase and lymphocyte kinase; preferably anucleic acid molecule, in particular a recombinant DNA molecule orcloned gene, encoding the alpha kinase has a nucleotide sequence or iscomplementary to a DNA sequence as set forth in any of SEQ ID NOS: 26,28, 30, 32, 34, 36, 38 and 40.

The murine and/or human DNA sequences of the alpha kinase genes of thepresent invention or portions thereof, may be prepared as probes toscreen for complementary sequences and genomic clones in the same oralternate species. The present invention extends to probes so preparedthat may be provided for screening cDNA and genomic libraries for thealpha kinase genes. For example, the probes may be prepared with avariety of known vectors, such as the phage λ vector. The presentinvention also includes the preparation of plasmids including suchvectors, and the use of the DNA sequences to construct vectorsexpressing antisense RNA or ribozymes which would attack the mRNAs ofany or all of the DNA sequences set forth in any of SEQ ID NOS: 26, 28,30, 32, 34, 36, 38 and 40. Correspondingly, the preparation of antisenseRNA and ribozymes are included herein.

According to other preferred features of certain preferred embodimentsof the present invention, a recombinant expression system is provided toproduce biologically active animal or human alpha kinase selected fromthe group of melanoma kinase, heart kinase, kidney kinase, skeletalmuscle kinase and lymphocyte kinase.

The present invention naturally contemplates several means forpreparation of the alpha kinase of the present invention, including asillustrated herein known recombinant techniques, and the invention isaccordingly intended to cover such synthetic preparations within itsscope. The isolation of the cDNA and amino acid sequences disclosedherein facilitates the production of the alpha kinase of the presentinvention by such recombinant techniques, and accordingly, the inventionextends to expression vectors prepared from the disclosed DNA sequencesfor expression in host systems by recombinant DNA techniques, and to theresulting transformed hosts.

In a further aspect, the invention provides a recombinant DNA expressionvector comprising the nucleic acid encoding an alpha kinase proteinselected from the group of melanoma alpha kinase, kidney alpha kinase,heart alpha kinase, skeletal muscle alpha kinase and lymphocyte alphakinase, wherein the DNA encoding the alpha kinase is operativelyassociated with an expression control sequence. The invention alsoprovides a transformed host cell transfected with said DNA vector.

The invention further includes a unicellular host transformed with arecombinant DNA molecule comprising a DNA sequence or degenerate variantthereof, which encodes an alpha kinase, or a fragment thereof, selectedfrom the group consisting of:

-   -   a. the DNA sequence of (SEQ ID NO: 26);    -   b. the DNA sequence of (SEQ ID NO: 28);    -   c. the DNA sequence of (SEQ ID NO: 30);    -   d. the DNA sequence of (SEQ ID NO: 32);    -   e. the DNA sequence of (SEQ ID NO: 34);    -   d. the DNA sequence of (SEQ ID NO: 36);    -   e. the DNA sequence of (SEQ ID NO: 38);    -   h. the DNA sequence of (SEQ ID NO: 40);    -   i. DNA sequences that hybridize to any of the foregoing DNA        sequences under standard hybridization conditions; and    -   j. DNA sequences that code on expression for an amino acid        sequence encoded by any of the foregoing DNA sequences;    -   wherein said DNA sequence is operatively linked to an expression        control sequence.

Such a unicellular host is particularly selected from the groupconsisting of E. coli, Pseudomonas, Bacillus, Streptomyces, yeasts, CHO,R1.1, B-W, L-M, COS 1, COS 7, BSC1, BSC40, and BMT10 cells, plant cells,insect cells, mouse cells and human cells in tissue culture.

In a further aspect, the present invention includes an isolated proteincharacterized by the presence of at least two domains, one of thedomains being an alpha-kinase catalytic domain and the other domainbeing an ion channel domain.

Thus, the present invention provides an isolated melanoma alpha kinaseprotein characterized by having an alpha-kinase catalytic domain and anion channel domain. In particular, a melanoma alpha kinase protein isprovided which comprises the amino acid sequence set out in SEQ ID NO:27 and 29, and analogs, variants and fragments thereof. The inventionprovides a melanoma alpha kinase protein which comprises the amino acidsequence set out in SEQ ID NO: 27 or 29, and variants thereof whereinone or more amino acids is substituted with a conserved amino acid.

The invention further provides an isolated kidney alpha kinase proteincharacterized by having an alpha-kinase catalytic domain and an ionchannel domain. In particular, the kidney alpha kinase protein comprisesthe amino acid sequence set out in SEQ ID NO: 31 and 33, and analogs,variants and fragments thereof. The invention provides a kidney alphakinase protein which comprises the amino acid sequence set out in SEQ IDNO: 31 or 33, and variants thereof wherein one or more amino acids issubstituted with a conserved amino acid.

The present invention further provides an isolated heart alpha kinaseprotein. In particular, the heart alpha kinase protein comprises theamino acid sequence set out in SEQ ID NO: 35 and 37, and analogs,variants and immunogenic fragments thereof. The invention provides aheart alpha kinase protein which comprises the amino acid sequence setout in SEQ ID NO: 35 or 37, and variants thereof wherein one or moreamino acids is substituted with a conserved amino acid.

The present invention still further provides an isolated skeletal musclealpha kinase protein. In particular, the skeletal muscle alpha kinaseprotein comprises the amino acid sequence set out in SEQ ID NO: 39, andanalogs, variants and immunogenic fragments thereof. The inventionprovides a skeletal muscle alpha kinase protein which comprises theamino acid sequence set out in SEQ ID NO: 39, and variants thereofwherein one or more amino acids is substituted with a conserved aminoacid.

The invention includes an isolated lymphocyte alpha kinase protein. Inparticular, the lymphocyte alpha kinase protein comprises the amino acidsequence set out in SEQ ID NO: 41, and analogs, variants and immunogenicfragments thereof. The invention provides a lymphocyte alpha kinaseprotein which comprises the amino acid sequence set out in SEQ ID NO:41, and variants thereof wherein one or more amino acids is substitutedwith a conserved amino acid.

In a particular aspect, the present invention includes a pharmaceuticalcomposition comprising one or more alpha kinase protein selected fromthe group of melanoma alpha kinase, kidney alpha kinase, heart alphakinase, skeletal muscle alpha kinase and lymphocyte alpha kinase, and apharmaceutically acceptable carrier.

In a further aspect, the invention provides a purified antibody to analpha kinase protein selected from the group of melanoma alpha kinase,kidney alpha kinase, heart alpha kinase, skeletal muscle alpha kinaseand lymphocyte alpha kinase.

A monoclonal antibody to an alpha kinase protein selected from the groupof melanoma alpha kinase, kidney alpha kinase, heart alpha kinase,skeletal muscle alpha kinase and lymphocyte alpha kinase is stillfurther provided. The invention includes an immortal cell line thatproduces a monoclonal antibody to an alpha kinase protein selected fromthe group of melanoma alpha kinase, kidney alpha kinase, heart alphakinase, skeletal muscle alpha kinase and lymphocyte alpha kinase.

Any such contemplated antibody may be labeled with a detectable label.The label may be selected from the group consisting of an enzyme, achemical which fluoresces, and a radioactive element.

The invention further includes an antibody to an alpha kinase proteinselected from the group of melanoma alpha kinase, kidney alpha kinase,heart alpha kinase, skeletal muscle alpha kinase and lymphocyte alphakinase, which recognizes the phosphorylated form of the alpha kinase ora phosphorylated fragment thereof.

The present invention likewise extends to antibodies againstspecifically phosphorylated alpha kinase targets, including naturallyraised and recombinantly prepared antibodies. These antibodies and theirlabeled counterparts are included within the scope of the presentinvention for their particular ability in detecting alpha kinaseactivity via detection of the phosphorylated product by ELISA or anyother immunoassay known to the skilled artisan.

In the instance where a radioactive label, such as the isotopes ³H, ¹⁴C,³²P, ³³p ³⁵S, ³⁶Cl, ⁵¹Cr, ⁵⁷Co, ⁵⁸Co, ⁵⁹Fe, ⁹⁰Y, ¹²⁵I, ¹³¹I, and ¹⁸⁶Reare used, known currently available counting procedures may be utilized.In the instance where the label is an enzyme, detection may beaccomplished by any of the presently utilized calorimetric,spectrophotometric, fluorospectrophotometric, amperometric or gasometrictechniques known in the art.

The present invention provides a method for treating an animal in needof increased activity of melanoma alpha kinase which comprisesadministration of melanoma alpha kinase to the animal.

The present invention further provides a method for treating an animalin need of increased activity of melanoma alpha kinase which comprisesadministration of an antibody against melanoma alpha kinase to theanimal.

The present invention also provides a method for treating an animal inneed of increased activity of kidney alpha kinase which comprisesadministration of kidney alpha kinase to the animal.

The invention also includes a method for treating an animal in need ofincreased activity of kidney alpha kinase which comprises administrationof an antibody against kidney alpha kinase to the animal.

The invention further provides a method for treating an animal in needof increased activity of heart alpha kinase which comprisesadministration of heart alpha kinase to the animal.

The present invention also contemplates a method for treating an animalin need of increased activity of heart alpha kinase which comprisesadministration of an antibody against heart alpha kinase to the animal.

In an additional aspect, the invention provides a method for treating ananimal in need of increased activity of skeletal muscle alpha kinasewhich comprises administration of skeletal muscle alpha kinase to theanimal.

A method for treating an animal in need of increased activity ofskeletal muscle alpha kinase which comprises administration of anantibody against skeletal muscle alpha kinase to the animal is furtherprovided.

The present invention includes method for treating an animal in need ofincreased activity of lymphocyte alpha kinase which comprisesadministration of lymphocyte alpha kinase to the animal.

The present invention further provides a method for treating an animalin need of increased activity of lymphocyte alpha kinase which comprisesadministration of an antibody against lymphocyte alpha kinase to theanimal.

The therapeutic method provided herein could include the method for thetreatment of various pathologies or other cellular dysfunctions andderangements by the administration of pharmaceutical compositions thatmay comprise effective inhibitors of alpha kinase activity, or otherequally effective drugs developed for instance by a drug screening assayprepared and used in accordance with a further aspect of the presentinvention.

The invention includes an assay system for screening of potential drugseffective at attenuating alpha kinase activity of target mammalian cellsby interrupting or potentiating the phosphorylation of alpha kinaseselected from the group of melanoma kinase, heart kinase, kidney kinase,skeletal muscle kinase and lymphocyte kinase. In one instance, the testdrug could be administered to a cellular sample along with ATP carryinga detectable label on its γ-phosphate that gets transferred to thekinase target, including the kinase itself, or a peptide substrate, bythe particular alpha kinase. Quantification of the labeled kinase targetor peptide substrate is diagnostic of the candidate drug's efficacy. Afurther embodiment would provide for the assay to be performed using apurely in vitro system comprised of the alpha kinase, ATP or labeledATP, the kinase target or peptide substrate, appropriate buffer, anddetection reagents and/or instrumentation to detect and quantify theextent of alpha kinase-directed phosphorylation activity.

The assay system could more importantly be adapted to identify drugs orother entities that are capable of binding to the alpha kinase and/orits cognate phosphorylation target, either in the cytoplasm or in thenucleus, thereby inhibiting or potentiating alpha kinase activity andits resultant phenotypic outcome. Such an assay would be useful in thedevelopment of drugs that would be specific against particular cellularactivity, or that would potentiate such activity, in time or in level ofactivity. For example, such drugs might be used to treat variouscarcinomas or other hyperproliferative pathologies.

In an additional aspect, the present invention includes a method fordetecting the presence or activity of an alpha kinase protein selectedfrom the group of melanoma alpha kinase, kidney alpha kinase, heartalpha kinase, skeletal muscle alpha kinase and lymphocyte alpha kinase,wherein said alpha kinase is measured by:

A. contacting a biological sample from a mammal in which the presence oractivity of said alpha kinase is suspected with a binding partner ofsaid alpha kinase under conditions that allow binding of said alphakinase to said binding partner to occur; and

B. detecting whether binding has occurred between said alpha kinase fromsaid sample and the binding partner;

wherein the detection of binding indicates that presence or activity ofsaid alpha kinase in said sample.

The present invention further provides a method for detecting thepresence of an alpha kinase protein selected from the group of melanomaalpha kinase, kidney alpha kinase, heart alpha kinase, skeletal musclealpha kinase and lymphocyte alpha kinase, wherein the alpha kinase ismeasured by:

-   -   a. contacting a sample in which the presence or activity of an        alpha kinase protein selected from the group of melanoma alpha        kinase, kidney alpha kinase, heart alpha kinase, skeletal muscle        alpha kinase and lymphocyte alpha kinase is suspected with an        antibody to the said alpha kinase protein under conditions that        allow binding of the alpha kinase protein to the binding partner        to occur; and    -   b. detecting whether binding has occurred between the alpha        kinase protein from the sample and the antibody;        wherein the detection of binding indicates the presence or        activity of the alpha kinase protein in the sample.

In a still further aspect, the invention provides a method of testingthe ability of a drug or other entity to modulate the kinase activity ofan alpha kinase protein selected from the group of melanoma alphakinase, kidney alpha kinase, heart alpha kinase, skeletal muscle alphakinase and lymphocyte alpha kinase which comprises:

A. culturing a colony of test cells containing the alpha kinase protein;

B. adding the drug or other entity under test; and

C. measuring the kinase activity of said alpha kinase protein in thetest cells, wherein when the amount of kinase activity in the presenceof the modulator is greater than in its absence, the modulator isidentified as an agonist or activator of the alpha kinase protein,whereas when the amount of kinase activity in the presence of themodulator is less than in its absence, the modulator is identified as anantagonist or inhibitor of the alpha kinase protein.

Other objects and advantages will become apparent to those skilled inthe art from a review of the ensuing description which proceeds withreference to the following illustrative drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and B. A, Sequence alignment of the catalytic domains of humaneEF-2 kinase, C. elegans eEF-2 kinase, MHCK A, MHCK B and clone FC-ANO9.Identical amino acids (bold) and conserved hydrophobic amino acids (°)are noted. B, Phylogenetic tree of sequences shown in (A), with theaddition of mouse and rat eEF-2 kinases. Tree was obtained using the J.Hein method with PAM250 residue weight table. The following accessionnumbers were used for the sequences: U93846–U93850, 1495779, 1170675,1903458, C22986.

FIG. 2 depicts a sequence alignment of C. elegans, mouse, human eEF-2kinase, and the catalytic domain of Dictyostelium discoideum MHCK A.Identical amino acids are indicated by dark blue boxed regions andchemically conserved amino acids are indicated by light blue shadedregions. Amino acids in the human sequence that are identical to themouse sequence are represented by dots. Amino acids underlined in blackcorrespond to the six regions that match peptides obtained from thesequencing of purified rabbit reticulocyte eEF-2 kinase. The GXGXXGnucleotide-binding motif is underlined in red. The blue dashed line overresidues 625–632 in C. elegans eEF-2 kinases designates the amino acidscorresponding to exon 4, which is missing in Cefk-2.

FIG. 3 depicts a schematic representation of the structure of mammalianand C. elegans eEF-2 kinases and MHCK A. The homologous regions arerepresented by dark shading. The regions of weak similarity arerepresented by light shading. The position of the GXGXXG (SEQ ID NO: 21)motif is indicated by vertical arrows.

FIG. 4 depicts a sequence alignment of C. elegans, mouse, human eEF-2kinase, and the catalytic domain of Dictyostelium discoideum MHCK A,heart kinase, melanoma kinase and ch4 kinase. Identical amino acids areindicated by dark blue boxed regions and chemically conserved aminoacids are indicated by light blue shaded regions.

FIGS. 5A through C depicts the nucleic acid sequence of mouse melanomaalpha-kinase (MK).

FIGS. 6A and B depicts the predicted amino acid sequence of mousemelanoma alpha-kinase (MK).

FIGS. 7A and B depicts the nucleic acid sequence (A) and predicted aminoacid sequence (B) of human melanoma alpha-kinase (MK).

FIGS. 8A and B depicts the nucleic acid sequence (A) and predicted aminoacid sequence (B) of human heart alpha-kinase (HK).

FIGS. 9A and B depicts the nucleic acid sequence (A) and predicted aminoacid sequence (B) of human kidney alpha-kinase (KK).

FIGS. 10A and B depicts the nucleic acid sequence (A) and predictedamino acid sequence (B) of human skeletal muscle alpha-kinase (SK).

FIGS. 11A and B depicts the nucleic acid sequence (A) and predictedamino acid sequence (B) of human lymphocyte alpha-kinase (LK).

FIG. 12 shows the alignment of the catalytic domains of the clonedalpha-kinases.

FIG. 13 depicts a phylogeneic analysis of the cloned alpha-kinases.

FIG. 14 shows the time course of ³²P incorporation into expressedmaltose-binding protein-melanoma alpha-kinase fusion protein (MBP-MK).

FIGS. 15A through F shows Northern Blot analysis of the tissuedistribution of the alpha-kinases in human and mouse tissues. StandardMultiple Tisue Northern (MTN) blots (Clontech) were stained as describedin Materials and Methods. A, B, C: Blots probed for Melanoma Kinase; A:Human MTN Blot B: Human Immune System MTN Blot II. C: Mouse MTN Blot. D:Human 12-Lane MTN Blot probed for Kidney kinase. E: Human 12-Lane MTNBlot probed for Muscle kinase. F: Mouse MTN Blot probed for Heartkinase. (abbreviations: sk. muscle—skeletal muscle, p.b.leukocyte—peripheral bood leukocyte, s. intestine—small intestine).

FIG. 16 shows a comparison of the ion channel portions of melanomakinase (MK), kidney kinase (KK) and melastatin (ME).

FIG. 17 Sequence alignment of MK and KK with members of the LTRP channelsubfamily. Roman numerals designate the six predicted transmembranesegments. Black boxes highlight identical amino acids. Gray boxeshighlight conserved amino acids. The alignment was constructed using theClustalW program and the shading was done using the Boxshade program.

FIG. 18. Schematic representation of five new α-kinases described inthis paper together with eEF-2 kinase and the Dictyostelium MHCKs.

FIGS. 19. A and B. Phylogenetic tree of the LTRP channel subfamily. Thistree was generated from the full-length protein sequences using theClustalW program. B. The proposed structural model of MK and KK.

DETAILED DESCRIPTION

Protein phosphorylation plays a pivotal role in a wide variety ofcellular processes. Enzymes which assist in protein phosphorylation arereferred to as “protein kinases.” Two protein kinase superfamilies havebeen described. The vast majority of protein kinases belong to theserine/threonine/tyrosine kinase superfamily. Several hundred members ofthis superfamily have thus far been characterized and found to sharesimilar structural organization of their catalytic domains consisting of12 conserved subdomains. There is also the histidine kinase superfamilyconsisting primarily of sensor components of the prokaryotictwo-component signal transduction systems. Eukaryotic members of thissuperfamily have been recently described. In addition, mitochondrialbranched-chain-ketoacid dehydrogenase kinase and the mitochondrialpyruvate dehydrogenase kinase have been described which are structurallyrelated to the histidine kinases, but phosphorylate their substrates onserine. The existence of several protein kinases have recently beenreported which have very little or no homology to either superfamily.This new superfamily is termed alpha-kinase. The first two members eEF-2kinase and MHCKA kinase differ from conventional protein kinases in thatthey phosphorylate amino acids located within α-helices. Thus, inaddition to the two well-characterized superfamily of eukaryotic proteinkinases, which phosphorylate amino acids located in loops and turns,there appears to be a third superfamily of α-helix-directed kinases.

Additional novel members of the alpha kinase superfamily have hereinbeen cloned and sequenced. In particular, these new alphakinases—melanoma alpha kinase, kidney alpha kinase, heart alpha kinase,skeletal muscle alpha kinase and lymphocyte kinase—represent new membersof the alpha kinase superfamily. The alpha kinases of the presentinvention are related to eEF-2 kinase and MHCK A and join the alphakinase superfamily. In addition, however, the novel alpha kinases of thepresent invention have new and unique characteristics.

In particular, the melanoma alpha kinase and kidney kinase of thepresent invention have a unique structure. These proteins have twodomains, one domain is the alpha-kinase catalytic domain and the otheris an ion channel. This is the first recognized example of an ionchannel being covalently linked to a protein kinase. It is likely thatthese novel protein kinases can be regulated by ion flow through themembrane. Expression of the melanoma kinase was detected in all mousetissues studied, including heart, skeletal muscle, brain, liver andlung. This kinase is the most abundant in the heart. In contrast, thekidney kinase is present almost exclusively in kidney tissue. The ionchannel portion is very similar to (70% identical) to a previouslyidentified protein called melastatin that is selectively downregulatedin metastatic tumors, and therefore is believed to be a metastasissuppressor gene. Melanoma alpha-kinase, kidney alpha-kinase, as well asmelastatin, belong to the TRP family of ion channels. All TRP proteinsfunction as tetramers, and various TRP proteins can form tetramers indifferent combinations that result in ion channels with differentproperties. Considering the high degree of similarity between melanomakinase, kidney kinase, and melastatin, it is likely that melanoma kinaseand kidney kinase can form tetrameric complexes with melastatin.

In accordance with the present invention there may be employedconventional molecular biology, microbiology, and recombinant DNAtechniques within the skill of the art. Such techniques are explainedfully in the literature. See, e.g., Sambrook et al, “Molecular Cloning:A Laboratory Manual” (1989); “Current Protocols in Molecular Biology”Volumes I–III [Ausubel, R. M., ed. (1994)]; “Cell Biology: A LaboratoryHandbook” Volumes I–III [J. E. Celis, ed. (1994))]; “Current Protocolsin Immunology” Volumes I–III [Coligan, J. E., ed. (1994)];“Oligonucleotide Synthesis” (M. J. Gait ed. 1984); “Nucleic AcidHybridization” [B. D. Hames & S. J. Higgins eds. (1985)]; “TranscriptionAnd Translation” [B. D. Hames & S. J. Higgins, eds. (1984)]; “AnimalCell Culture” [R. I. Freshney, ed. (1986)]; “Immobilized Cells AndEnzymes” [IRL Press, (1986)]; B. Perbal, “A Practical Guide To MolecularCloning” (1984).

Therefore, if appearing herein, the following terms shall have thedefinitions set out below.

The terms “elongation factor-2 kinase”, “eEF-2 kinase”, “EF-2 kinase”,“Cefk”, and any variants not specifically listed, may be used hereininterchangeably, and as used throughout the present application andclaims refer to proteinaceous material including single or multipleproteins, and extends to those proteins having the amino acid sequencedata described herein and presented in FIGS. 1 and 5 (SEQ ID NOS: 1, 2,6, 8 and 14), and the profile of activities set forth herein and in theclaims. Accordingly, proteins displaying substantially equivalent oraltered activity are likewise contemplated. These modifications may bedeliberate, for example, such as modifications obtained throughsite-directed mutagenesis, or may be accidental, such as those obtainedthrough mutations in hosts that are producers of the complex or itsnamed subunits. Also, the terms elongation factor-2 kinase”, “eEF-2kinase”, “EF-2 kinase”, and “Cefk” are intended to include within theirscope proteins specifically recited herein as well as all substantiallyhomologous analogs and allelic variations. The terms “melanoma αkinase”, “melanoma alpha kinase ”, “melanoma kinase”, “MK”, and anyvariants not specifically listed, may be used herein interchangeably,and as used throughout the present application and claims refer toproteinaceous material including single or multiple proteins, andextends to those proteins having the amino acid sequence data describedherein and presented in FIGS. 6 and 7 (SEQ ID NOS: 27 and 29), and theprofile of activities set forth herein and in the claims. Accordingly,proteins displaying substantially equivalent or altered activity arelikewise contemplated. These modifications may be deliberate, forexample, such as modifications obtained through site-directedmutagenesis, or may be accidental, such as those obtained throughmutations in hosts that are producers of the complex or its namedsubunits. Also, the terms “melanoma α kinase”, “melanoma alpha kinase”,“melanoma kinase” and “MK” are intended to include within their scopeproteins specifically recited herein as well as all substantiallyhomologous analogs and allelic variations.

The terms “heart α kinase”, “heart alpha kinase”, “heart kinase”, “HK”,and any variants not specifically listed, may be used hereininterchangeably, and as used throughout the present application andclaims refer to proteinaceous material including single or multipleproteins, and extends to those proteins having the amino acid sequencedata described herein and presented in FIG. 8 (SEQ ID NOS: 35 and 37),and the profile of activities set forth herein and in the claims.Accordingly, proteins displaying substantially equivalent or alteredactivity are likewise contemplated. These modifications may bedeliberate, for example, such as modifications obtained throughsite-directed mutagenesis, or may be accidental, such as those obtainedthrough mutations in hosts that are producers of the complex or itsnamed subunits. Also, the terms “heart α kinase”, “heart alpha kinase ”,“heart kinase”, “HK” are intended to include within their scope proteinsspecifically recited herein as well as all substantially homologousanalogs and allelic variations.

The terms “kidney α kinase”, “kidney alpha kinase”, “kidney kinase”,“KK”, and any variants not specifically listed, may be used hereininterchangeably, and as used throughout the present application andclaims refer to proteinaceous material including single or multipleproteins, and extends to those proteins having the amino acid sequencedata described herein and presented in FIG. 9 (SEQ ID NOS: 31 and 33),and the profile of activities set forth herein and in the claims.Accordingly, proteins displaying substantially equivalent or alteredactivity are likewise contemplated. These modifications may bedeliberate, for example, such as modifications obtained throughsite-directed mutagenesis, or may be accidental, such as those obtainedthrough mutations in hosts that are producers of the complex or itsnamed subunits. Also, the terms “kidney α kinase”, “kidney alphakinase”, “kidney kinase”, “KK” are intended to include within theirscope proteins specifically recited herein as well as all substantiallyhomologous analogs and allelic variations.

The terms “skeletal muscle α kinase”, “skeletal muscle alpha kinase”,“skeletal muscle kinase”, “SK”, and any variants not specificallylisted, may be used herein interchangeably, and as used throughout thepresent application and claims refer to proteinaceous material includingsingle or multiple proteins, and extends to those proteins having theamino acid sequence data described herein and presented in FIG. 10 (SEQID NO: 39), and the profile of activities set forth herein and in theclaims. Accordingly, proteins displaying substantially equivalent oraltered activity are likewise contemplated. These modifications may bedeliberate, for example, such as modifications obtained throughsite-directed mutagenesis, or may be accidental, such as those obtainedthrough mutations in hosts that are producers of the complex or itsnamed subunits. Also, the terms “skeletal muscle α kinaes”, “skeletalmuscle alpha kinase”, “skeletal muscle kinase”, “SK” are intended toinclude within their scope proteins specifically recited herein as wellas all substantially homologous analogs and allelic variations.

The terms “lymphocyte α kinase”, “lymphocyte alpha kinase”, “lymphocytekinase”, “LK”, “Ch4” and any variants not specifically listed, may beused herein interchangeably, and as used throughout the presentapplication and claims refer to proteinaceous material including singleor multiple proteins, and extends to those proteins having the aminoacid sequence data described herein and presented in FIG. 11 (SEQ ID NO:41), and the profile of activities set forth herein and in the claims.Accordingly, proteins displaying substantially equivalent or alteredactivity are likewise contemplated. These modifications may bedeliberate, for example, such as modifications obtained throughsite-directed mutagenesis, or may be accidental, such as those obtainedthrough mutations in hosts that are producers of the complex or itsnamed subunits. Also, the terms “lymphocyte α kinaes”, “lymphocyte alphakinase”, “lymphocyte kinase”, “LK”, “Ch4” are intended to include withintheir scope proteins specifically recited herein as well as allsubstantially homologous analogs and allelic variations.

The amino acid residues described herein are preferred to be in the “L”isomeric form. However, residues in the “D” isomeric form can besubstituted for any L-amino acid residue, as long as the desiredfractional property of immunoglobulin-binding is retained by thepolypeptide. NH₂ refers to the free amino group present at the aminoterminus of a polypeptide. COOH refers to the free carboxy group presentat the carboxy terminus of a polypeptide. In keeping with standardpolypeptide nomenclature, J. Biol. Chem., 243:3552–59 (1969),abbreviations for amino acid residues are shown in the following Tableof Correspondence:

TABLE OF CORRESPONDENCE SYMBOL 1-Letter 3-Letter AMINO ACID Y Tyrtyrosine G Gly glycine F Phe phenylalanine M Met methionine A Alaalanine S Ser serine I Ile isoleucine L Leu leucine T Thr threonine VVal valine P Pro proline K Lys lysine H His histidine Q Gln glutamine EGlu glutamic acid W Trp tryptophan R Arg arginine D Asp aspartic acid NAsn asparagine C Cys cysteine

It should be noted that all amino-acid residue sequences are representedherein by formulae whose left and right orientation is in theconventional direction of amino-terminus to carboxy-terminus.Furthermore, it should be noted that a dash at the beginning or end ofan amino acid residue sequence indicates a peptide bond to a furthersequence of one or more amino-acid residues. The above Table ispresented to correlate the three-letter and one-letter notations whichmay appear alternately herein.

Nucleic Acids

The present invention provides an isolated nucleic acid encodingmelanoma alpha kinase, or a fragment thereof having at least 15nucleotides. In particular, the invention provides an isolated nucleicacid encoding human melanoma alpha kinase, wherein the nucleic acid isselected from the group consisting of:

-   -   a. the DNA sequence of SEQ ID NO: 26;    -   b. DNA sequences that hybridize to the sequence of subpart (a)        under moderate stringency hybridization conditions;    -   c. DNA sequences capable of encoding the amino acid sequence        encoded by the DNA sequences of (a) or (b);    -   d. degenerate variants thereof;    -   e. alleles thereof; and    -   f. hybridizable fragments thereof.

In particular, the invention provides an isolated nucleic acid encodingmouse melanoma alpha kinase, wherein the nucleic acid is selected fromthe group consisting of:

-   -   a. the DNA sequence of SEQ ID NO: 28;    -   b. DNA sequences that hybridize to the sequence of subpart (a)        under moderate stringency hybridization conditions;    -   c. DNA sequences capable of encoding the amino acid sequence        encoded by the DNA sequences of (a) or (b);    -   d. degenerate variants thereof;    -   e. alleles thereof; and    -   f. hybridizable fragments thereof.

The present invention further provides an isolated nucleic acid encodingheart alpha kinase, or a fragment thereof having at least 15nucleotides. In particular, the present invention provides an isolatednucleic acid encoding human heart alpha kinase, wherein the nucleic acidis selected from the group consisting of:

-   -   a. nucleic acid comprising the DNA sequence of SEQ ID NO: 34;    -   b. DNA sequences that hybridize to the sequence of subpart (a)        under moderate stringency hybridization conditions;    -   c. DNA sequences capable of encoding the amino acid sequence        encoded by the DNA sequences of (a) or (b);    -   d. degenerate variants thereof;    -   e. alleles thereof; and    -   f. hybridizable fragments thereof.

In particular, the present invention provides an isolated nucleic acidencoding mouse heart alpha kinase, wherein the nucleic acid is selectedfrom the group consisting of:

-   -   a. nucleic acid comprising the DNA sequence of SEQ ID NO: 36;    -   b. DNA sequences that hybridize to the sequence of subpart (a)        under moderate stringency hybridization conditions;    -   c. DNA sequences capable of encoding the amino acid sequence        encoded by the DNA sequences of (a) or (b);    -   d. degenerate variants thereof;    -   e. alleles thereof; and    -   f. hybridizable fragments thereof.

The present invention still further provides an isolated nucleic acidencoding kidney alpha kinase, or a fragment thereof having at least 15nucleotides. In particular, the invention includes an isolated nucleicacid encoding human kidney alpha kinase, wherein the nucleic acid isselected from the group consisting of:

-   -   a. the DNA sequence of SEQ ID NO: 30;    -   b. DNA sequences that hybridize to the sequence of subpart (a)        under moderate stringency hybridization conditions;    -   c. DNA sequences capable of encoding the amino acid sequence        encoded by the DNA sequences of (a) or (b);    -   d. degenerate variants thereof;    -   e. alleles thereof; and    -   f. hybridizable fragments thereof.

In particular, the invention includes an isolated nucleic acid encodingmouse kidney alpha kinase, wherein the nucleic acid is selected from thegroup consisting of:

-   -   a. the DNA sequence of SEQ ID NO: 32;    -   b. DNA sequences that hybridize to the sequence of subpart (a)        under moderate stringency hybridization conditions;    -   c. DNA sequences capable of encoding the amino acid sequence        encoded by the DNA sequences of (a) or (b);    -   d. degenerate variants thereof;    -   e. alleles thereof; and    -   f. hybridizable fragments thereof.

The present invention also provides an isolated nucleic acid encodingskeletal muscle alpha kinase, or a fragment thereof having at least 15nucleotides. In particular, an isolated nucleic acid encoding skeletalmuscle alpha kinase is provided, wherein the nucleic acid is selectedfrom the group consisting of:

-   -   a. nucleic acid comprising the DNA sequence of SEQ ID NO: 38;    -   b. DNA sequences that hybridize to the sequence of subpart (a)        under moderate stringency hybridization conditions;    -   c. DNA sequences capable of encoding the amino acid sequence        encoded by the DNA sequences of (a) or (b);    -   d. degenerate variants thereof;    -   e. alleles thereof; and    -   f. hybridizable fragments thereof.

The present invention also includes an isolated nucleic acid encodinglymphocyte alpha kinase, or a fragment thereof having at least 15nucleotides. In particular, the present invention provides an isolatednucleic acid encoding lymphocyte alpha kinase, wherein the nucleic acidis selected from the group consisting of:

-   -   a. nucleic acid comprising the DNA sequence of SEQ ID NO: 40;    -   b. DNA sequences that hybridize to the sequence of subpart (a)        under moderate stringency hybridization conditions;    -   c. DNA sequences capable of encoding the amino acid sequence        encoded by the DNA sequences of (a) or (b);    -   d. degenerate variants thereof;    -   e. alleles thereof, and    -   f. hybridizable fragments thereof.

The present invention also relates to a recombinant DNA molecule orcloned gene, or a degenerate variant thereof, which encodes an alphakinase selected from the group of melanoma kinase, heart kinase, kidneykinase, skeletal muscle kinase and lymphocyte kinase; preferably anucleic acid molecule, in particular a recombinant DNA molecule orcloned gene, encoding the alpha kinase has a nucleotide sequence or iscomplementary to a DNA sequence as set forth in any of SEQ ID NOS: 26,28, 30, 32, 34, 36, 38 and 40.

The murine and/or human DNA sequences of the alpha kinase genes of thepresent invention or portions thereof, may be prepared as probes toscreen for complementary sequences and genomic clones in the same oralternate species. The present invention extends to probes so preparedthat may be provided for screening cDNA and genomic libraries for thealpha kinase genes. For example, the probes may be prepared with avariety of known vectors, such as the phage λ vector. The presentinvention also includes the preparation of plasmids including suchvectors, and the use of the DNA sequences to construct vectorsexpressing antisense RNA or ribozymes which would attack the mRNAs ofany or all of the DNA sequences set forth in any of SEQ ID NOS: 26, 28,30, 32, 34, 36, 38 and 40. Correspondingly, the preparation of antisenseRNA and ribozymes are included herein.

According to other preferred features of certain preferred embodimentsof the present invention, a recombinant expression system is provided toproduce biologically active animal or human alpha kinase selected fromthe group of melanoma kinase, heart kinase, kidney kinase, skeletalmuscle kinase and lymphocyte kinase.

The present invention naturally contemplates several means forpreparation of the alpha kinase of the present invention, including asillustrated herein known recombinant techniques, and the invention isaccordingly intended to cover such synthetic preparations within itsscope. The isolation of the cDNA and amino acid sequences disclosedherein facilitates the production of the alpha kinase of the presentinvention by such recombinant techniques, and accordingly, the inventionextends to expression vectors prepared from the disclosed DNA sequencesfor expression in host systems by recombinant DNA techniques, and to theresulting transformed hosts.

In a further aspect, the invention provides a recombinant DNA expressionvector comprising the nucleic acid encoding an alpha kinase proteinselected from the group of melanoma alpha kinase, kidney alpha kinase,heart alpha kinase, skeletal muscle alpha kinase and lymphocyte alphakinase, wherein the DNA encoding the alpha kinase is operativelyassociated with an expression control sequence. The invention alsoprovides a transformed host cell transfected with said DNA vector.

The invention further includes a unicellular host transformed with arecombinant DNA molecule comprising a DNA sequence or degenerate variantthereof, which encodes an alpha kinase, or a fragment thereof, selectedfrom the group consisting of:

-   -   a. the DNA sequence of (SEQ ID NO: 26);    -   b. the DNA sequence of (SEQ ID NO: 28);    -   c. the DNA sequence of (SEQ ID NO: 30);    -   d. the DNA sequence of (SEQ ID NO: 32);    -   e. the DNA sequence of (SEQ ID NO: 34);    -   f. the DNA sequence of (SEQ ID NO: 36);    -   g. the DNA sequence of (SEQ ID NO: 38);    -   h. the DNA sequence of (SEQ ID NO: 40);    -   i. DNA sequences that hybridize to any of the foregoing DNA        sequences under standard hybridization conditions; and    -   j. DNA sequences that code on expression for an amino acid        sequence encoded by any of the foregoing DNA sequences;

wherein said DNA sequence is operatively linked to an expression controlsequence.

Such a unicellular host is particularly selected from the groupconsisting of E. coli, Pseudomonas, Bacillus, Streptomyces, yeasts, CHO,R1.1, B-W, L-M, COS 1, COS 7, BSC1, BSC40, and BMT10 cells, plant cells,insect cells, mouse cells and human cells in tissue culture.

A “replicon” is any genetic element (e.g., plasmid, chromosome, virus)that functions as an autonomous unit of DNA replication in vivo; i.e.,capable of replication under its own control.

A “vector” is a replicon, such as plasmid, phage or cosmid, to whichanother DNA segment may be attached so as to bring about the replicationof the attached segment.

A “DNA molecule” refers to the polymeric form of deoxyribonucleotides(adenine, guanine, thymine, or cytosine) in its either single strandedform, or a double-stranded helix. This term refers only to the primaryand secondary structure of the molecule, and does not limit it to anyparticular tertiary forms. Thus, this term includes double-stranded DNAfound, inter alia, in linear DNA molecules (e.g., restrictionfragments), viruses, plasmids, and chromosomes. In discussing thestructure of particular double-stranded DNA molecules, sequences may bedescribed herein according to the normal convention of giving only thesequence in the 5′ to 3′ direction along the nontranscribed strand ofDNA (i.e., the strand having a sequence homologous to the mRNA).

An “origin of replication” refers to those DNA sequences thatparticipate in DNA synthesis.

A DNA “coding sequence” is a double-stranded DNA sequence which istranscribed and translated into a polypeptide in vivo when placed underthe control of appropriate regulatory sequences. The boundaries of thecoding sequence are determined by a start codon at the 5′ (amino)terminus and a translation stop codon at the 3′ (carboxyl) terminus. Acoding sequence can include, but is not limited to, prokaryoticsequences, cDNA from eukaryotic mRNA, genomic DNA sequences fromeukaryotic (e.g., mammalian) DNA, and even synthetic DNA sequences. Apolyadenylation signal and transcription termination sequence willusually be located 3′ to the coding sequence.

Transcriptional and translational control sequences are DNA regulatorysequences, such as promoters, enhancers, polyadenylation signals,terminators, and the like, that provide for the expression of a codingsequence in a host cell.

A “promoter sequence” is a DNA regulatory region capable of binding RNApolymerase in a cell and initiating transcription of a downstream (3′direction) coding sequence. For purposes of defining the presentinvention, the promoter sequence is bounded at its 3′ terminus by thetranscription initiation site and extends upstream (5′ direction) toinclude the minimum number of bases or elements necessary to initiatetranscription at levels detectable above background. Within the promotersequence will be found a transcription initiation site (convenientlydefined by mapping with nuclease S1), as well as protein binding domains(consensus sequences) responsible for the binding of RNA polymerase.Eukaryotic promoters will often, but not always, contain “TATA” boxesand “CAT” boxes. Prokaryotic promoters contain Shine-Dalgamo sequencesin addition to the −10 and −35 consensus sequences.

An “expression control sequence” is a DNA sequence that controls andregulates the transcription and translation of another DNA sequence. Acoding sequence is “under the control” of transcriptional andtranslational control sequences in a cell when RNA polymerasetranscribes the coding sequence into mRNA, which is then translated intothe protein encoded by the coding sequence.

A DNA sequence is “operatively linked” to an expression control sequencewhen the expression control sequence controls and regulates thetranscription and translation of that DNA sequence. The term“operatively linked” includes having an appropriate start signal (e.g.,ATG) in front of the DNA sequence to be expressed and maintaining thecorrect reading frame to permit expression of the DNA sequence under thecontrol of the expression control sequence and production of the desiredproduct encoded by the DNA sequence. If a gene that one desires toinsert into a recombinant DNA molecule does not contain an appropriatestart signal, such a start signal can be inserted in front of the gene.

The term “standard hybridization conditions” refers to salt andtemperature conditions substantially equivalent to 5×SSC and 65° C. forboth hybridization and wash. However, one skilled in the art willappreciate that such “standard hybridization conditions” are dependenton particular conditions including the concentration of sodium andmagnesium in the buffer, nucleotide sequence length and concentration,percent mismatch, percent formamide, and the like. Also important in thedetermination of “standard hybridization conditions” is whether the twosequences hybridizing are RNA—RNA, DNA—DNA or RNA-DNA. Such standardhybridization conditions are easily determined by one skilled in the artaccording to well known formulae, wherein hybridization is typically10–20° C. below the predicted or determined T_(m) with washes of higherstringency, if desired.

In one aspect, the present invention relates to the identification of anew superfamily of protein kinases, denoted alpha kinases. Accordingly,it includes the DNA sequences coding for these family members. Inaddition, the invention also contemplates that each member of this newprotein kinase superfamily has its own cognate phosphorylation target.

A “signal sequence” can be included before the coding sequence. Thissequence encodes a signal peptide, N-terminal to the polypeptide, thatcommunicates to the host cell to direct the polypeptide to the cellsurface or secrete the polypeptide into the media, and this signalpeptide is clipped off by the host cell before the protein leaves thecell. Signal sequences can be found associated with a variety ofproteins native to prokaryotes and eukaryotes.

The term “oligonucleotide,” as used herein in referring to the probe ofthe present invention, is defined as a molecule comprised of two or moreribonucleotides, preferably more than three. Its exact size will dependupon many factors which, in turn, depend upon the ultimate function anduse of the oligonucleotide.

The term “primer” as used herein refers to an oligonucleotide, whetheroccurring naturally as in a purified restriction digest or producedsynthetically, which is capable of acting as a point of initiation ofsynthesis when placed under conditions in which synthesis of a primerextension product, which is complementary to a nucleic acid strand, isinduced, i.e., in the presence of nucleotides and an inducing agent suchas a DNA polymerase and at a suitable temperature and pH. The primer maybe either single-stranded or double-stranded and must be sufficientlylong to prime the synthesis of the desired extension product in thepresence of the inducing agent. The exact length of the primer willdepend upon many factors, including temperature, source of primer anduse of the method. For example, for diagnostic applications, dependingon the complexity of the target sequence, the oligonucleotide primertypically contains 15–25 or more nucleotides, although it may containfewer nucleotides.

The primers herein are selected to be “substantially” complementary todifferent strands of a particular target DNA sequence. This means thatthe primers must be sufficiently complementary to hybridize with theirrespective strands. Therefore, the primer sequence need not reflect theexact sequence of the template. For example, a non-complementarynucleotide fragment may be attached to the 5′ end of the primer, withthe remainder of the primer sequence being complementary to the strand.

Alternatively, non-complementary bases or longer sequences can beinterspersed into the primer, provided that the primer sequence hassufficient complementarity with the sequence of the strand to hybridizetherewith and thereby form the template for the synthesis of theextension product.

A “heterologous” region of the DNA construct is an identifiable segmentof DNA within a larger DNA molecule that is not found in associationwith the larger molecule in nature. Thus, when the heterologous regionencodes a mammalian gene, the gene will usually be flanked by DNA thatdoes not flank the mammalian genomic DNA in the genome of the sourceorganism. Another example of a heterologous coding sequence is aconstruct where the coding sequence itself is not found in nature (e.g.,a cDNA where the genomic coding sequence contains introns, or syntheticsequences having codons different than the native gene). Allelicvariations or naturally-occurring mutational events do not give rise toa heterologous region of DNA as defined herein.

As used herein, the terms “restriction endonucleases” and “restrictionenzymes” refer to bacterial enzymes, each of which cut double-strandedDNA at or near a specific nucleotide sequence.

A cell has been “transformed” by exogenous or heterologous DNA when suchDNA has been introduced inside the cell. The transforming DNA may or maynot be integrated (covalently linked) into chromosomal DNA making up thegenome of the cell. In prokaryotes, yeast, and mammalian cells forexample, the transforming DNA may be maintained on an episomal elementsuch as a plasmid. With respect to eukaryotic cells, a stablytransformed cell is one in which the transforming DNA has becomeintegrated into a chromosome so that it is inherited by daughter cellsthrough chromosome replication. This stability is demonstrated by theability of the eukaryotic cell to establish cell lines or clonescomprised of a population of daughter cells containing the transformingDNA. A “clone” is a population of cells derived from a single cell orcommon ancestor by mitosis. A “cell line” is a clone of a primary cellthat is capable of stable growth in vitro for many generations.

Two DNA sequences are “substantially homologous” when at least about 75%(preferably at least about 80%, and most preferably at least about 90 or95%) of the nucleotides match over the defined length of the DNAsequences. Sequences that are substantially homologous can be identifiedby comparing the sequences using standard software available in sequencedata banks, or in a Southern hybridization experiment under, forexample, stringent conditions as defined for that particular system.Defining appropriate hybridization conditions is within the skill of theart. See, e.g., Maniatis et al., supra; DNA Cloning, Vols. I & II,supra; Nucleic Acid Hybridization, supra.

It should be appreciated that also within the scope of the presentinvention are DNA sequences encoding alpha kinase, selected from thegroup of melanoma kinase, kidney kinase, heart kinase, skeletal musclekinase and lymphocyte kinase, which code for a protein having the sameamino acid sequence as any of SEQ ID NOS:, but which are degenerate toany of SEQ ID NOS:. By “degenerate to” is meant that a differentthree-letter codon is used to specify a particular amino acid. It iswell known in the art that the following codons can be usedinterchangeably to code for each specific amino acid:

Phenylalanine (Phe or F) UUU or UUC Leucine (Leu or L) UUA or UUG or CUUor CUC or CUA or CUG Isoleucine (Ile or I) AUU or AUC or AUA Methionine(Met or M) AUG Valine (Val or V) GUU or GUC of GUA or GUG Serine (Ser orS) UCU or UCC or UCA or UCG or AGU or AGC Proline (Pro or P) CCU or CCCor CCA or CCG Threonine (Thr or T) ACU or ACC or ACA or ACG Alanine (Alaor A) GCU or GCG or GCA or GCG Tyrosine (Tyr or Y) UAU or UAC Histidine(His or H) CAU or CAC Glutamine (Gln or Q) CAA or CAG Asparagine (Asn orN) AAU or AAC Lysine (Lys or K) AAA or AAG Aspartic Acid (Asp or D) GAUor GAC Glutamic Acid (Glu or E) GAA or GAG Cysteine (Cys or C) UGU orUGC Arginine (Arg or R) CGU or CGC or CGA or CGG or AGA or AGG Glycine(Gly or G) GGU or GGC or GGA or GGG Tryptophan (Trp or W) UGGTermination codon UAA (ochre) or UAG (amber) or UGA (opal)

It should be understood that the codons specified above are for RNAsequences. The corresponding codons for DNA have a T substituted for U.

Mutations can be made in any of SEQ ID NOS: such that a particular codonis changed to a codon which codes for a different amino acid. Such amutation is generally made by making the fewest nucleotide changespossible. A substitution mutation of this sort can be made to change anamino acid in the resulting protein in a non-conservative manner (i.e.,by changing the codon from an amino acid belonging to a grouping ofamino acids having a particular size or characteristic to an amino acidbelonging to another grouping) or in a conservative manner (i.e., bychanging the codon from an amino acid belonging to a grouping of aminoacids having a particular size or characteristic to an amino acidbelonging to the same grouping). Such a conservative change generallyleads to less change in the structure and function of the resultingprotein. A non-conservative change is more likely to alter thestructure, activity or function of the resulting protein. The presentinvention should be considered to include sequences containingconservative changes which do not significantly alter the activity orbinding characteristics of the resulting protein.

The following is one example of various groupings of amino acids: (I)Amino acids with nonpolar R groups: Alanine, Valine, Leucine,Isoleucine, Proline, Phenylalanine, Tryptophan, Methionine; (II) Aminoacids with uncharged polar R groups: Glycine, Serine, Threonine,Cysteine, Tyrosine, Asparagine, Glutamine; (III) Amino acids withcharged polar R groups (negatively charged at pH 6.0): Aspartic acid,Glutamic acid; (IV) Basic amino acids (positively charged at pH 6.0):Lysine, Arginine, Histidine (at pH 6.0).

Another grouping may be those amino acids with phenyl groups:Phenylalanine, Tryptophan, Tyrosine

Another grouping may be according to molecular weight (i.e., size of Rgroups):

Glycine  75 Alanine  89 Serine 105 Proline 115 Valine 117 Threonine 119Cysteine 121 Leucine 131 Isoleucine 131 Asparagine 132 Aspartic acid 133Glutamine 146 Lysine 146 Glutamic acid 147 Methionine 149 Histidine (atpH 6.0) 155 Phenylalanine 165 Arginine 174 Tyrosine 181 Tryptophan 204

Particularly preferred substitutions are:

-   Lys for Arg and vice versa such that a positive charge may be    maintained;-   Glu for Asp and vice versa such that a negative charge may be    maintained;-   Ser for Thr such that a free —OH can be maintained; and-   Gln for Asn such that a free NH₂ can be maintained.

Amino acid substitutions may also be introduced to substitute an aminoacid with a particularly preferable property. For example, a Cys may beintroduced a potential site for disulfide bridges with another Cys. AHis may be introduced as a particularly “catalytic” site (i.e., His canact as an acid or base and is the most common amino acid in biochemicalcatalysis). Pro may be introduced because of its particularly planarstructure, which induces β-turns in the protein's structure.

Another feature of this invention is the expression of the DNA sequencesdisclosed herein. As is well known in the art, DNA sequences may beexpressed by operatively linking them to an expression control sequencein an appropriate expression vector and employing that expression vectorto transform an appropriate unicellular host.

Such operative linking of a DNA sequence of this invention to anexpression control sequence, of course, includes, if not already part ofthe DNA sequence, the provision of an initiation codon, ATG, in thecorrect reading frame upstream of the DNA sequence.

A wide variety of host/expression vector combinations may be employed inexpressing the DNA sequences of this invention. Useful expressionvectors, for example, may consist of segments of chromosomal,non-chromosomal and synthetic DNA sequences. Suitable vectors includederivatives of SV40 and known bacterial plasmids, e.g., E. coli plasmidscol E1, pCR1, pBR322, pMB9 and their derivatives, plasmids such as RP4;phage DNAS, e.g., the numerous derivatives of phage λ, e.g., NM989, andother phage DNA, e.g., M13 and filamentous single stranded phage DNA;yeast plasmids such as the 2μ plasmid or derivatives thereof; vectorsuseful in eukaryotic cells, such as vectors useful in insect ormammalian cells; vectors derived from combinations of plasmids and phageDNAs, such as plasmids that have been modified to employ phage DNA orother expression control sequences; and the like.

Any of a wide variety of expression control sequences—sequences thatcontrol the expression of a DNA sequence operatively linked to it—may beused in these vectors to express the DNA sequences of this invention.Such useful expression control sequences include, for example, the earlyor late promoters of SV40, CMV, vaccinia, polyoma or adenovirus, the lacsystem, the trp system, the TAC system, the TRC system, the LTR system,the major operator and promoter regions of phage λ, the control regionsof fd coat protein, the promoter for 3-phosphoglycerate kinase or otherglycolytic enzymes, the promoters of acid phosphatase (e.g., Pho5), thepromoters of the yeast α-mating factors, and other sequences known tocontrol the expression of genes of prokaryotic or eukaryotic cells ortheir viruses, and various combinations thereof.

A wide variety of unicellular host cells are also useful in expressingthe DNA sequences of this invention. These hosts may include well knowneukaryotic and prokaryotic hosts, such as strains of E. coli,Pseudomonas, Bacillus, Streptomyces, fungi such as yeasts, and animalcells, such as CHO, R1.1, B-W and L-M cells, African Green Monkey kidneycells (e.g., COS 1, COS 7, BSC1, BSC40, and BMT10), insect cells (e.g.,Sf9), and human cells and plant cells in tissue culture.

It will be understood that not all vectors, expression control sequencesand hosts will function equally well to express the DNA sequences ofthis invention. Neither will all hosts function equally well with thesame expression system. However, one skilled in the art will be able toselect the proper vectors, expression control sequences, and hostswithout undue experimentation to accomplish the desired expressionwithout departing from the scope of this invention. For example, inselecting a vector, the host must be considered because the vector mustfunction in it. The vector's copy number, the ability to control thatcopy number, and the expression of any other proteins encoded by thevector, such as antibiotic markers, will also be considered.

In selecting an expression control sequence, a variety of factors willnormally be considered. These include, for example, the relativestrength of the system, its controllability, and its compatibility withthe particular DNA sequence or gene to be expressed, particularly asregards potential secondary structures. Suitable unicellular hosts willbe selected by consideration of, e.g., their compatibility with thechosen vector, their secretion characteristics, their ability to foldproteins correctly, and their fermentation requirements, as well as thetoxicity to the host of the product encoded by the DNA sequences to beexpressed, and the ease of purification of the expression products.

Considering these and other factors, a person skilled in the art will beable to construct a variety of vector/expression control sequence/hostcombinations that will express the DNA sequences of this invention onfermentation or in large scale animal culture.

The present invention extends to the preparation of antisenseoligonucleotides and ribozymes that may be used to interfere with theexpression of the eEF-2 kinase gene at the translational level. Thisapproach utilizes antisense nucleic acid and ribozymes to blocktranslation of a specific mRNA, either by masking that mRNA with anantisense nucleic acid or cleaving it with a ribozyme.

Antisense nucleic acids are DNA or RNA molecules that are complementaryto at least a portion of a specific mRNA molecule. (See Weintraub, 1990;Marcus-Sekura, 1988.) In the cell, they hybridize to that mRNA, forminga double stranded molecule. The cell does not translate an mRNA in thisdouble-stranded form. Therefore, antisense nucleic acids interfere withthe expression of mRNA into protein. Oligomers of about fifteennucleotides and molecules that hybridize to the AUG initiation codonwill be particularly efficient, since they are easy to synthesize andare likely to pose fewer problems than larger molecules when introducingthem into eEF-2 kinase-producing cells. Antisense methods have been usedto inhibit the expression of many genes in vitro (Marcus-Sekura, 1988;Hambor et al., 1988).

Ribozymes are RNA molecules possessing the ability to specificallycleave other single stranded RNA molecules in a manner somewhatanalogous to DNA restriction endonucleases. Ribozymes were discoveredfrom the observation that certain mRNAs have the ability to excise theirown introns. By modifying the nucleotide sequence of these RNAs,researchers have been able to engineer molecules that recognize specificnucleotide sequences in an RNA molecule and cleave it (Cech, 1988.).Because they are sequence-specific, only mRNAs with particular sequencesare inactivated.

Investigators have identified two types of ribozymes, Tetrahymena-typeand “hammerhead”-type. (Hasselhoff and Gerlach, 1988) Tetrahymena-typeribozymes recognize four-base sequences, while “hammerhead”-typerecognize eleven- to eighteen-base sequences. The longer the recognitionsequence, the more likely it is to occur exclusively in the target mRNAspecies. Therefore, hammerhead-type ribozymes are preferable toTetrahymena-type ribozymes for inactivating a specific mRNA species, andeighteen base recognition sequences are preferable to shorterrecognition sequences.

Polypeptides

In a further aspect, the present invention includes an isolated proteincharacterized by the presence of at least two domains, one of thedomains being an alpha-kinase catalytic domain and the other domainbeing an ion channel domain.

Thus, the present invention provides an isolated melanoma alpha kinaseprotein characterized by having an alpha-kinase catalytic domain and anion channel domain. In particular, a melanoma alpha kinase protein isprovided which comprises the amino acid sequence set out in SEQ ID NO:27 and 29, and analogs, variants and fragments thereof.

The invention further provides an isolated kidney alpha kinase proteincharacterized by having an alpha-kinase catalytic domain and an ionchannel domain. In particular, the kidney alpha kinase protein comprisesthe amino acid sequence set out in SEQ ID NO: 31 and 33, and analogs,variants and fragments thereof.

The present invention further provides an isolated heart alpha kinaseprotein. In particular, the heart alpha kinase protein comprises theamino acid sequence set out in SEQ ID NO: 35 and 37, and analogs,variants and immunogenic fragments thereof.

The present invention still further provides an isolated skeletal musclealpha kinase protein. In particular, the skeletal muscle alpha kinaseprotein comprises the amino acid sequence set out in SEQ ID NO: 39, andanalogs, variants and immunogenic fragments thereof.

The invention includes an isolated lymphocyte alpha kinase protein. Inparticular, the lymphocyte alpha kinase protein comprises the amino acidsequence set out in SEQ ID NO: 41, and analogs, variants and immunogenicfragments thereof.

Two amino acid sequences are “substantially homologous” when at leastabout 70% of the amino acid residues (preferably at least about 80%, andmost preferably at least about 90 or 95%) are identical, or representconservative substitutions.

Antibodies

In a further aspect, the invention provides a purified antibody to analpha kinase protein selected from the group of melanoma alpha kinase,kidney alpha kinase, heart alpha kinase, skeletal muscle alpha kinaseand lymphocyte alpha kinase.

A monoclonal antibody to an alpha kinase protein selected from the groupof melanoma alpha kinase, kidney alpha kinase, heart alpha kinase,skeletal muscle alpha kinase and lymphocyte alpha kinase is stillfurther provided. the invention includes an immortal cell line thatproduces a monoclonal antibody to an alpha kinase protein selected fromthe group of melanoma alpha kinase, kidney alpha kinase, heart alphakinase, skeletal muscle alpha kinase and lymphocyte alpha kinase.

Any such contemplated antibody may be labeled with a detectable label.The label may be selected from the group consisting of an enzyme, achemical which fluoresces, and a radioactive element.

The invention further includes an antibody to an alpha kinase proteinselected from the group of melanoma alpha kinase, kidney alpha kinase,heart alpha kinase, skeletal muscle alpha kinase and lymphocyte alphakinase, which recognizes the phosphorylated form of the alpha kinase ora phosphorylated fragment thereof.

The present invention likewise extends to antibodies againstspecifically phosphorylated alpha kinase targets, including naturallyraised and recombinantly prepared antibodies. These antibodies and theirlabeled counterparts are included within the scope of the presentinvention for their particular ability in detecting alpha kinaseactivity via detection of the phosphorylated product by ELISA or anyother immunoassay known to the skilled artisan.

In the instance where a radioactive label, such as the isotopes ³H, ¹⁴C,³²P, ³³P, ³⁵S, ³⁶Cl, ⁵¹Cr, ⁵⁷Co, ⁵⁸Co, ⁵⁹Fe, ⁹⁰Y, ¹²⁵I, ¹³¹I, and ¹⁸⁶Reare used, known currently available counting procedures may be utilized.In the instance where the label is an enzyme, detection may beaccomplished by any of the presently utilized colorimetric,spectrophotometric, fluorospectrophotometric, amperometric or gasometrictechniques known in the art.

An “antibody” is any immunoglobulin, including antibodies and fragmentsthereof, that binds a specific epitope. The term encompasses polyclonal,monoclonal, and chimeric antibodies, the last mentioned described infurther detail in U.S. Pat. Nos. 4,816,397 and 4,816,567.

An “antibody combining site” is that structural portion of an antibodymolecule comprised of heavy and light chain variable and hypervariableregions that specifically binds antigen.

The phrase “antibody molecule” in its various grammatical forms as usedherein contemplates both an intact immunoglobulin molecule and animmunologically active portion of an immunoglobulin molecule.

Exemplary antibody molecules are intact immunoglobulin molecules,substantially intact immunoglobulin molecules and those portions of animmunoglobulin molecule that contains the paratope, including thoseportions known in the art as Fab, Fab′, F(ab′)₂ and F(v), which portionsare preferred for use in the therapeutic methods described herein.

Fab and F(ab′)₂ portions of antibody molecules are prepared by theproteolytic reaction of papain and pepsin, respectively, onsubstantially intact antibody molecules by methods that are well-known.See for example, U.S. Pat. No. 4,342,566 to Theofilopolous et al. Fab′antibody molecule portions are also well-known and are produced fromF(ab′)₂ portions followed by reduction of the disulfide bonds linkingthe two heavy chain portions as with mercaptoethanol, and followed byalkylation of the resulting protein mercaptan with a reagent such asiodoacetamide. An antibody containing intact antibody molecules ispreferred herein.

The phrase “monoclonal antibody” in its various grammatical forms refersto an antibody having only one species of antibody combining sitecapable of immunoreacting with a particular antigen. A monoclonalantibody thus typically displays a single binding affinity for anyantigen with which it immunoreacts. A monoclonal antibody may thereforecontain an antibody molecule having a plurality of antibody combiningsites, each immunospecific for a different antigen; e.g., a bispecific(chimeric) monoclonal antibody.

In a particular embodiment, the present invention relates tophosphorylation target analogs, which are short peptide sequencesderived from phosphorylation targets of this new superfamily of proteinalpha kinases centered around the alpha kinases selected from the groupof melanoma kinase, kidney kinase, heart kinase, skeletal muscle kinaseand lymphocyte kinase. Specifically, it is contemplated that thesepeptide analogs will be instrumental in the development of highthroughput screening assays to identify inhibitors of members of thisnew superfamily.

Also, antibodies including both polyclonal and monoclonal antibodies,and drugs that modulate the production or activity of alpha kinase maypossess certain diagnostic applications and may, for example, beutilized for the purpose of detecting and/or measuring levels of alphakinase. It is anticipated that further experimentation will reveal aprognostic correlation between alpha kinase levels and the predictionand or progression of certain malignancies associated with carcinoma.For example, alpha kinase may be used to produce both polyclonal andmonoclonal antibodies to themselves in a variety of cellular media, byknown techniques such as the hybridoma technique utilizing, for example,fused mouse spleen lymphocytes and myeloma cells.

Likewise, small molecules that mimic or antagonize the activity of alphakinase of the invention may be discovered or synthesized, and may beused in diagnostic and/or therapeutic protocols.

The general methodology for making monoclonal antibodies by hybridomasis well known. Immortal, antibody-producing cell lines can also becreated by techniques other than fusion, such as direct transformationof B lymphocytes with oncogenic DNA, or transfection with Epstein-Barrvirus. See, e.g., M. Schreier et al., “Hybridoma Techniques” (1980);Hammerling et al., “Monoclonal Antibodies And T-cell Hybridomas” (1981);Kennett et al., “Monoclonal Antibodies” (1980); see also U.S. Pat. Nos.4,341,761; 4,399,121; 4,427,783; 4,444,887; 4,451,570; 4,466,917;4,472,500; 4,491,632; 4,493,890.

Panels of monoclonal antibodies produced against alpha kinase peptidescan be screened for various properties; i.e., isotype, epitope,affinity, etc. Of particular interest are monoclonal antibodies thatneutralize the activity of alpha kinase. Such monoclonals can be readilyidentified in alpha kinase activity assays. High affinity antibodies arealso useful when immunoaffinity purification of native or recombinantalpha kinase is desired.

Preferably, the anti-alpha kinase antibody used in the diagnosticmethods of this invention is an affinity purified polyclonal antibody.More preferably, the antibody is a monoclonal antibody (mAb). Inaddition, it is preferable for the anti-alpha kinase antibody moleculesused herein be in the form of Fab, Fab′, F(ab′)₂ or F(v) portions ofwhole antibody molecules.

As suggested earlier, the diagnostic method of the present inventioncomprises examining a cellular sample or medium by means of an assayincluding an effective amount of an antagonist to alpha kinase, such asan anti-alpha kinase antibody, preferably an affinity-purifiedpolyclonal antibody, and more preferably a mAb. In addition, it ispreferable for the anti-alpha kinase antibody molecules used herein bein the form of Fab, Fab′, F(ab′)₂ or F(v) portions or whole antibodymolecules. As previously discussed, patients capable of benefitting fromthis method include those suffering from cancer, a pre-cancerous lesion,a viral infection or other like pathological derangement. Methods forisolating the alpha kinase and inducing anti-alpha kinase antibodies andfor determining and optimizing the ability of anti-alpha kinaseantibodies to assist in the examination of the target cells are allwell-known in the art.

Methods for producing polyclonal anti-polypeptide antibodies arewell-known in the art. See U.S. Pat. No. 4,493,795 to Nestor et al. Amonoclonal antibody, typically containing Fab and/or F(ab′)₂ portions ofuseful antibody molecules, can be prepared using the hybridomatechnology described in Antibodies—A Laboratory Manual, Harlow and Lane,eds., Cold Spring Harbor Laboratory, New York (1988), which isincorporated herein by reference.

Splenocytes are typically fused with myeloma cells using polyethyleneglycol (PEG) 6000. Fused hybrids are selected by their sensitivity toHAT. Hybridomas producing a monoclonal antibody useful in practicingthis invention are identified by their ability to immunoreact with aparticular kinase and of the present invention and their ability toinhibit specified alpha kinase activity in target cells.

A monoclonal antibody useful in practicing the present invention can beproduced by initiating a monoclonal hybridoma culture comprising anutrient medium containing a hybridoma that secretes antibody moleculesof the appropriate antigen specificity. The culture is maintained underconditions and for a time period sufficient for the hybridoma to secretethe antibody molecules into the medium. The antibody-containing mediumis then collected. The antibody molecules can then be further isolatedby well-known techniques.

Media useful for the preparation of these compositions are bothwell-known in the art and commercially available and include syntheticculture media, inbred mice and the like. An exemplary synthetic mediumis Dulbecco's minimal essential medium (DMEM; Dulbecco et al., Virol.8:396 (1959)) supplemented with 4.5 gm/l glucose, 20 mM glutamine, and20% fetal calf serum. An exemplary inbred mouse strain is the Balb/c.

Methods for producing monoclonal anti-alpha kinase antibodies are alsowell-known in the art. See Niman et al., Proc. Natl. Acad. Sci. USA,80:4949–4953 (1983). Typically, the present alpha kinase or a peptideanalog is used either alone or conjugated to an immunogenic carrier, asthe immunogen in the before described procedure for producing anti-alphakinase monoclonal antibodies. The hybridomas are screened for theability to produce an antibody that immunoreacts with the eEF-2 kinasepeptide analog and the present alpha kinase.

Therapeutic Compositions and Methods

Therapeutic possibilities are raised by the knowledge of the alphakinase sequences, melanoma kinase, kidney kinase, heart kinase, skeletalmuscle kinase and lymphocyte kinase. Accordingly, it is contemplatedthat sequences that are derived from the complement to the alpha kinasemRNA sequence, and various modifications thereof, can act as potentantisense drugs that either inhibit expression in a competitive fashion,or, more effectively, by nuclease activity associated with the antisensedrug that cleaves the alpha kinase mRNA sequence, thus rendering itirreversibly inactive. Alternative therapeutics are also contemplatedthat concern the use of peptides and peptide analogs representingportions of phosphorylation target amino acid sequences. It isenvisioned that such peptide-based drugs would inhibit alpha kinaseactivity on its native target, thus bypassing the cascade of events thatwould lead to malignant transformation.

In a particular aspect, the present invention includes a pharmaceuticalcomposition comprising one or more alpha kinase protein selected fromthe group of melanoma alpha kinase, kidney alpha kinase, heart alphakinase, skeletal muscle alpha kinase and lymphocyte alpha kinase, and apharmaceutically acceptable carrier.

The present invention provides a method for treating an animal in needof increased activity of melanoma alpha kinase which comprisesadministration of melanoma alpha kinase to the animal.

The present invention further provides a method for treating an animalin need of increased activity of melanoma alpha kinase which comprisesadministration of an antibody against melanoma alpha kinase to theanimal.

The present invention also provides a method for treating an animal inneed of increased activity of kidney alpha kinase which comprisesadministration of kidney alpha kinase to the animal.

The invention also includes a method for treating an animal in need ofincreased activity of kidney alpha kinase which comprises administrationof an antibody against kidney alpha kinase to the animal.

The invention further provides a method for treating an animal in needof increased activity of heart alpha kinase which comprisesadministration of heart alpha kinase to the animal.

The present invention also contemplates a method for treating an animalin need of increased activity of heart alpha kinase which comprisesadministration of an antibody against heart alpha kinase to the animal.

In an additional aspect, the invention provides a method for treating ananimal in need of increased activity of skeletal muscle alpha kinasewhich comprises administration of skeletal muscle alpha kinase to theanimal.

A method for treating an animal in need of increased activity ofskeletal muscle alpha kinase which comprises administration of anantibody against skeletal muscle alpha kinase to the animal is furtherprovided.

The present invention includes method for treating an animal in need ofincreased activity of lymphocyte alpha kinase which comprisesadministration of lymphocyte alpha kinase to the animal.

The present invention further provides a method for treating an animalin need of increased activity of lymphocyte alpha kinase which comprisesadministration of an antibody against lymphocyte alpha kinase to theanimal.

The therapeutic method provided herein could include the method for thetreatment of various pathologies or other cellular dysfunctions andderangements by the administration of pharmaceutical compositions thatmay comprise effective inhibitors of alpha kinase activity, or otherequally effective drugs developed for instance by a drug screening assayprepared and used in accordance with a further aspect of the presentinvention.

It is a still further object of the present invention to provide amethod for the treatment of mammals to control the amount or activity ofalpha kinase, so as to alter the adverse consequences of such presenceor activity, or where beneficial, to enhance such activity.

It is a still further object of the present invention to provide amethod for the treatment of mammals to control the amount or activity ofalpha kinase, so as to treat or avert the adverse consequences ofinvasive, spontaneous or idiopathic pathological states.

The present invention further contemplates therapeutic compositionsuseful in practicing the therapeutic methods of this invention. Asubject therapeutic composition includes, in admixture, apharmaceutically acceptable excipient (carrier) and one or more of ananti-alpha kinase antibody, peptide analog capable of competing forphosphorylation of target by alpha kinase, antisense drug against alphakinase mRNA, or any other compound that is found to inhibit alpha kinaseactivity. In a preferred embodiment, the composition comprises anantigen capable of modulating the activity of alpha kinase within atarget cell.

The preparation of therapeutic compositions which contain polypeptides,analogs or active fragments as active ingredients is well understood inthe art. Typically, such compositions are prepared as injectables,either as liquid solutions or suspensions, however, solid forms suitablefor solution in, or suspension in, liquid prior to injection can also beprepared. The preparation can also be emulsified. The active therapeuticingredient is often mixed with excipients which are pharmaceuticallyacceptable and compatible with the active ingredient. Suitableexcipients are, for example, water, saline, dextrose, glycerol, ethanol,or the like and combinations thereof. In addition, if desired, thecomposition can contain minor amounts of auxiliary substances such aswetting or emulsifying agents, pH buffering agents which enhance theeffectiveness of the active ingredient.

A polypeptide, analog or active fragment can be formulated into thetherapeutic composition as neutralized pharmaceutically acceptable saltforms. Pharmaceutically acceptable salts include the acid addition salts(formed with the free amino groups of the polypeptide or antibodymolecule) and which are formed with inorganic acids such as, forexample, hydrochloric or phosphoric acids, or such organic acids asacetic, oxalic, tartaric, mandelic, and the like. Salts formed from thefree carboxyl groups can also be derived from inorganic bases such as,for example, sodium, potassium, ammonium, calcium, or ferric hydroxides,and such organic bases as isopropylamine, trimethylamine, 2-ethylaminoethanol, histidine, procaine, and the like.

The therapeutic polypeptide-, analog- or active fragment-containingcompositions are conventionally administered intravenously, as byinjection of a unit dose, for example. The term “unit dose” when used inreference to a therapeutic composition of the present invention refersto physically discrete units suitable as unitary dosage for humans, eachunit containing a predetermined quantity of active material calculatedto produce the desired therapeutic effect in association with therequired diluent; i.e., carrier, or vehicle.

The compositions are administered in a manner compatible with the dosageformulation, and in a therapeutically effective amount. The quantity tobe administered depends on the subject to be treated, capacity of thesubject's immune system to utilize the active ingredient, and degree ofinhibition or neutralization of eEF-2 kinase activity desired. Preciseamounts of active ingredient required to be administered depend on thejudgment of the practitioner and are peculiar to each individual.However, suitable dosages may range from about 0.1 to 20, preferablyabout 0.5 to about 10, and more preferably one to several, milligrams ofactive ingredient per kilogram body weight of individual per day anddepend on the route of administration. Suitable regimes for initialadministration and booster shots are also variable, but are typified byan initial administration followed by repeated doses at one or more hourintervals by a subsequent injection or other administration.Alternatively, continuous intravenous infusion sufficient to maintainconcentrations of ten nanomolar to ten micromolar in the blood arecontemplated.

Formulations

Intravenous Formulation I Ingredient mg/ml cefotaxime 250.0 antibody,peptide, antisense drug, or other compound 10.0 dextrose USP 45.0 sodiumbisulfite USP 3.2 edetate disodium USP 0.1 water for injection q.s.a.d.1.0 ml

Intravenous Formulation II Ingredient mg/ml ampicillin 250.0 antibody,peptide, antisense drug, or other compound 10.0 sodium bisulfite USP 3.2disodium edetate USP 0.1 water for injection q.s.a.d. 1.0 ml

Intravenous Formulation III Ingredient mg/ml gentamicin (charged assulfate) 40.0 antibody, peptide, antisense drug, or other compound 10.0sodium bisulfite USP 3.2 disodium edetate USP 0.1 water for injectionq.s.a.d. 1.0 ml

Intravenous Formulation IV Ingredient mg/ml antibody, peptide, antisensedrug, or other compound 10.0 dextrose USP 45.0 sodium bisulfite USP 3.2edetate disodium USP 0.1 water for injection q.s.a.d. 1.0 ml

As used herein, “pg” means picogram, “ng” means nanogram, “ug” or “μg”mean microgram, “mg” means milligram, “ul” or “μl” mean microliter, “ml”means milliliter, “l” means liter.

The phrase “pharmaceutically acceptable” refers to molecular entitiesand compositions that are physiologically tolerable and do not typicallyproduce an allergic or similar untoward reaction, such as gastric upset,dizziness and the like, when administered to a human.

The phrase “therapeutically effective amount” is used herein to mean anamount sufficient to prevent, and preferably reduce by at least about 30percent, more preferably by at least 50 percent, most preferably by atleast 90 percent, a clinically significant change in the S phaseactivity of a target cellular mass, or other feature of pathology suchas for example, elevated blood pressure, fever or white cell count asmay attend its presence and activity.

Assays and Methods

The present invention also relates to a variety of diagnosticapplications, including methods for detecting and quantifying the levelsof alpha kinase. As mentioned earlier, alpha kinase can be used toproduce antibodies to itself by a variety of known techniques, and suchantibodies could then be isolated and utilized as in tests for thepresence and levels of alpha kinase activity in suspect target cells.

The invention includes an assay system for screening of potential drugseffective at attenuating alpha kinase activity of target mammalian cellsby interrupting or potentiating the phosphorylation of alpha kinaseselected from the group of melanoma kinase, heart kinase, kdiney kinase,skeletal muscle kinase and lymphocyte kinase. In one instance, the testdrug could be administered to a cellular sample along with ATP carryinga detectable label on its γ-phosphate that gets transferred to thekinase target, including the kinase itself, or a peptide substrate, bythe particular alpha kinase. Quantification of the labeled kinase targetor peptide substrate is diagnostic of the candidate drug's efficacy. Afurther embodiment would provide for the assay to be performed using apurely in vitro system comprised of the alpha kinase, ATP or labeledATP, the kinase target or peptide substrate, appropriate buffer, anddetection reagents and/or instrumentation to detect and quantify theextent of alpha kinase-directed phosphorylation activity.

The assay system could more importantly be adapted to identify drugs orother entities that are capable of binding to the alpha kinase and/orits cognate phosphorylation target, either in the cytoplasm or in thenucleus, thereby inhibiting or potentiating alpha kinase activity andits resultant phenotypic outcome. Such an assay would be useful in thedevelopment of drugs that would be specific against particular cellularactivity, or that would potentiate such activity, in time or in level ofactivity. For example, such drugs might be used to treat variouscarcinomas or other hyperproliferative pathologies.

In an additional aspect, the present invention includes a method fordetecting the presence or activity of an alpha kinase protein selectedfrom the group of melanoma alpha kinase, kidney alpha kinase, heartalpha kinase, skeletal muscle alpha kinase and lymphocyte alpha kinase,wherein said alpha kinase is measured by:

A. contacting a biological sample from a mammal in which the presence oractivity of said alpha kinase is suspected with a binding partner ofsaid alpha kinase under conditions that allow binding of said alphakinase to said binding partner to occur; and

B. detecting whether binding has occurred between said alpha kinase fromsaid sample and the binding partner;

wherein the detection of binding indicates that presence or activity ofsaid alpha kinase in said sample.

The present invention further provides a method for detecting thepresence of an alpha kinase protein selected from the group of melanomaalpha kinase, kidney alpha kinase, heart alpha kinase, skeletal musclealpha kinase and lymphocyte alpha kinase, wherein the alpha kinase ismeasured by:

-   -   a. contacting a sample in which the presence or activity of an        alpha kinase protein selected from the group of melanoma alpha        kinase, kidney alpha kinase, heart alpha kinase, skeletal muscle        alpha kinase and lymphocyte alpha kinase is suspected with an        antibody to the said alpha kinase protein under conditions that        allow binding of the alpha kinase protein to the binding partner        to occur; and    -   b. detecting whether binding has occurred between the alpha        kinase protein from the sample and the antibody;        wherein the detection of binding indicates the presence or        activity of the alpha kinase protein in the sample.

In a still further aspect, the invention provides a method of testingthe ability of a drug or other entity to modulate the kinase activity ofan alpha kinase protein selected from the group of melanoma alphakinase, kidney alpha kinase, heart alpha kinase, skeletal muscle alphakinase and lymphocyte alpha kinase which comprises:

A. culturing a colony of test cells containing the alpha kinase protein;

B. adding the drug or other entity under test; and

C. measuring the kinase activity of said alpha kinase protein in thetest cells, wherein when the amount of kinase activity in the presenceof the modulator is greater than in its absence, the modulator isidentified as an agonist or activator of the alpha kinase protein,whereas when the amount of kinase activity in the presence of themodulator is less than in its absence, the modulator is identified as anantagonist or inhibitor of the alpha kinase protein.

It is a further object of the present invention to provide a method fordetecting alpha kinase activity in mammals in which invasive,spontaneous, or idiopathic pathological states are suspected to bepresent.

As described in detail above, antibody(ies) to alpha kinase can beproduced and isolated by standard methods including the well knownhybridoma techniques. For convenience, the antibody(ies) to alpha kinasewill be referred to herein as Ab₁ and antibody(ies) raised in anotherspecies as Ab₂.

The presence and levels of alpha kinase in cells can be ascertained bythe usual immunological procedures applicable to such determinations. Anumber of useful procedures are known. Three such procedures which areespecially useful, utilize either alpha kinase labeled with a detectablelabel, antibody Ab₁ labeled with a detectable label, or antibody Ab₂labeled with a detectable label. The procedures may be summarized by thefollowing equations wherein the asterisk indicates that the particle islabeled, and “˜” stands for alpha kinase:

-   A. ˜*+Ab₁=˜*Ab₁-   B. ˜+Ab*=˜Ab₁*-   C. ˜+Ab₁+Ab₂*=˜Ab₁Ab₂*

The procedures and their application are all familiar to those skilledin the art and accordingly may be utilized within the scope of thepresent invention. The “competitive” procedure, Procedure A, isdescribed in U.S. Pat. Nos. 3,654,090 and 3,850,752. Procedure C, the“sandwich” procedure, is described in U.S. Pat. Nos. RE 31,006 and4,016,043. Still other procedures are known such as the “doubleantibody,” or “DASP” procedure.

In each instance, alpha kinase forms complexes with one or moreantibody(ies) or binding partners and one member of the complex islabeled with a detectable label. The fact that a complex has formed and,if desired, the amount thereof, can be determined by known methodsapplicable to the detection of labels.

It will be seen from the above, that a characteristic property of Ab₂ isthat it will react with Ab₁. This is because Ab₁ raised in one mammalianspecies has been used in another species as an antigen to raise theantibody Ab₂. For example, Ab₂ may be raised in goats using rabbitantibodies as antigens. Ab₂ therefore would be anti-rabbit antibodyraised in goats. For purposes of this description and claims, Ab₁ willbe referred to as a primary or anti-alpha kinase antibody, and Ab₂ willbe referred to as a secondary or anti-Ab₁ antibody.

The labels most commonly employed for these studies are radioactiveelements, enzymes, chemicals which fluoresce when exposed to ultravioletlight, and others.

A number of fluorescent materials are known and can be utilized aslabels. These include, for example, fluorescein, rhodamine, auramine,Texas Red, AMCA blue and Lucifer Yellow. A particular detecting materialis anti-rabbit antibody prepared in goats and conjugated withfluorescein through an isothiocyanate.

eEF-2 kinase can also be labeled with a radioactive element or with anenzyme. The radioactive label can be detected by any of the currentlyavailable counting procedures. The preferred isotope may be selectedfrom ³H, ¹⁴C, ³²P, ³³P, ³⁵s, ³⁶Cl, ⁵¹Cr, ⁵⁷Co, ⁵⁸Co, ⁵⁹Fe, ⁹⁰Y, ¹²⁵I,¹³¹I, and ¹⁸⁶Re.

Enzyme labels are likewise useful, and can be detected by any of thepresently utilized colorimetric, spectrophotometric,fluorospectrophotometric, amperometric or gasometric techniques. Theenzyme is conjugated to the selected particle by reaction with bridgingmolecules such as carbodiimides, diisocyanates, glutaraldehyde and thelike. Many enzymes which can be used in these procedures are known andcan be utilized. The preferred are peroxidase, β-glucuronidase,β-D-glucosidase, β-D-galactosidase, urease, glucose oxidase plusperoxidase and alkaline phosphatase. U.S. Pat. Nos. 3,654,090;3,850,752; and 4,016,043 are referred to by way of example for theirdisclosure of alternate labeling material and methods.

A particular assay system developed and utilized in accordance with thepresent invention, is known as a receptor assay. In a receptor assay,the material to be assayed is appropriately labeled and then certaincellular test colonies are inoculated with a quantity of both thelabeled and unlabeled material after which binding studies are conductedto determine the extent to which the labeled material binds to the cellreceptors. In this way, differences in affinity between materials can beascertained.

Accordingly, a purified quantity of the alpha kinase may be radiolabeledand combined, for example, with antibodies or other inhibitors thereto,after which binding studies would be carried out. Solutions would thenbe prepared that contain various quantities of labeled and unlabeleduncombined alpha kinase, and cell samples would then be inoculated andthereafter incubated. The resulting cell monolayers are then washed,solubilized and then counted in a gamma counter for a length of timesufficient to yield a standard error of <5%. These data are thensubjected to Scatchard analysis after which observations and conclusionsregarding material activity can be drawn. While the foregoing isexemplary, it illustrates the manner in which a receptor assay may beperformed and utilized, in the instance where the cellular bindingability of the assayed material may serve as a distinguishingcharacteristic.

In accordance with the above, an assay system for screening potentialdrugs effective to modulate the activity of alpha kinase may beprepared. The alpha kinase may be introduced into a test system, and theprospective drug may also be introduced into the resulting cell culture,and the culture thereafter examined to observe any changes in the alphakinase activity of the cells, due either to the addition of theprospective drug alone, or due to the effect of added quantities of theknown alpha kinase. Alternatively, these assays can be carried out in apurely in vitro fashion as discussed below.

The invention may be better understood by reference to the followingnon-limiting Examples, which are provided as exemplary of the invention.The following examples are presented in order to more fully illustratethe preferred embodiments of the invention and should in no way beconstrued, however, as limiting the broad scope of the invention.

EXAMPLE 1 Molecular Cloning of cDNAs Encoding C. Elegans, Mouse, Rat,and Human eEF-2 Kinases

eEF-2 kinase from rabbit reticulocyte lysate was purified as described(Hait et al., (1996) FEBS Lett. 397:55–60). Peptides were generated fromthe nitrocellulose-bound 103-kDa eEF-2 kinase protein by in situ trypticdigestion (Erdjument-Bromage et al., (1994) Protein Sci. 3:2435–2446)and fractionated by reverse-phase HPLC (Elicone et al., (1994) J.Chromatogr. 676:121–137) using a 1.0 mm Reliasil C18 column. Selectedpeak fraction were then analyzed by a combination of automated Edmansequencing and matrix-assisted laser-desorption time-of-flight massspectrometry (Erdjument-Bromage et al., (1994)). The peptide sequencesprovided an essential lead into the cloning of eEF-2 kinase from human,mouse, rat, and Caenorhabditis elegans.

To clone the cDNA for C. elegans eEF-2 kinase, oligonucleotide primerswere designed based on the amino and carboxy termini of the predictedgene product from F42A10.4. Reverse transcriptase-PCR (RT-PCR) wasperformed using these primers and total RNA from C. elegans. A singlePCR product of ˜2.3 kb was obtained and gel-purified using a gelextraction kit (Qiagen, Chatsworth, Calif.). The fragment was ligatedinto vector pCR2.1 using the TA cloning kit (Invitrogen, SorrentoValley, Calif.), and then transformed into Escherichia coli. Plasmid DNAwas purified, and restriction analysis used to verify the orientation ofthe coding sequence with respect to the T7 promoter. Two clones (Cefk-1and Cefk-2, C. elegans eEF-2 kinase isoforms 1 and 2) were chosen andsequenced using a Li-Cor (Lincoln, Nebr.) Long Read IR model 400LAutomated DNA Sequencer. Analysis revealed that the two clones wereidentical except for a deletion of 24 bp in Cefk-2 which corresponds toexon 4 and probably represents an alternatively spliced form.

To clone the mouse eEF-2 kinase, degenerate primers were designed basedon the amino acid sequence of two peptides from rabbit eEF-2 kinase(LTPQAFSHFTFER (SEQ ID NO: 15) and LANXYYEKAE (SEQ ID NO: 16)): primerA,CA(G/A)GC(C/G/T/A)TT(C/T)(T/A)(C/G)(T/CCA(C/T)TT(C/T)AC(C/G/T/A)TT(C/T)GA(G/A(C/A)G(SEQ ID NO: 17); and primer B,TC(C/G/T/A)GC(C/T)TT(C/T)TC(G/A)TA(G/A)TA(C/T)TT(G/A)TT(C/G/A/T)GC (SEQID NO: 18). RT-PCR was performed using primers A and B and poly(A)⁺ RNAfrom mouse spleen (CLONTECH). A single PCR product (˜1.6 kb) was clonedinto pCR2.1 (Invitrogen) and sequenced. Using sequence information formthese mouse eEF-2 kinase cDNA fragments, new primers were designed for5′ rapid amplification of cDNA ends (RACE) and 3′ RACE to obtainfull-length mouse eEF-2 kinase cDNA. 5′ RACE and 3′ RACE were performedusing Marathon-Ready mouse spleen cDNA (CLONTECH). This was carried outaccording to the manufacturer's instructions using the primers AP1 and C

(TACAATCAGCTGATGACCAGAACGCTC) (SEQ ID NO: 19) 5′antisense, or D(GGATTTGGACTGGACAAGAACCCCC) (SEQ ID NO: 20) 3′sense.

To clone rat eEF-2 kinases, PCR was performed on a rat PC12 cDNA librarycloned in λGT10 (CLONTECH) using primer B and vector primers. A 700-bpfragment was specifically amplified. The fragment was cloned into pCR2.1(Invitrogen) and sequenced. This 700-bp fragments was radiolabeled andused to probe the same PC12 cDNA library (600,000 plaques). Fourteenpositives were obtained in the initial screening. Five plaques werechosen for further analysis and sequencing based on insert sizes thatranged from 1.4 to 2.0 kb.

Recently, eEF-2 kinase from rabbit reticulocyte lysate was purified tonear homogeneity (Hait et al., (1996)). This enabled determination ofits partial amino acid sequence as noted above. Two peptide sequences(LTPQAFSHFTFER (SEQ ID NO: 15) and LANXYYEKAE (SEQ ID NO: 16)) werecompared with entries in a nonredundant database using the NationalCenter for Biotechnology Information BLAST program (Altschul et al.,(1990) J. Mol. Biol. 215:403–410). Matches were found with a C. eleganshypothetical protein (F42A10.4; GenBank accession number U10414). Thissequence was obtained from the C. elegans genome sequencing project andis located on chromosome III (Wilson et al., (1994) Nature 368:32–38).The 100% identity between the sequenced peptides and the C. elegansprotein, as well as the fact that the predicted molecular weight of theC. elegans protein is similar to that of eEF-2 kinase, suggested thatthis gene encoded eEF-2 kinase. We cloned the full-length cDNA by RT-PCRusing C. elegans total RNA. Several clones were isolated and sequenced.Cefk-1 has six of the predicted exons and encodes 768 amino acids.Cefk-2 represents an alternatively spliced form that has five exons; itis missing amino acids 625–632 that correspond to exon four. Cefk-1 andCefk-2 were found to have eEF-2 kinase activity when expressed incell-free system using a wheat germ extract coupledtranscription/translation system.

To determine the amino acid sequence of mammalian eEF-2 kinase, wecloned and sequenced the cDNA of mouse eEF-2 kinase. We reasoned thatsince the sequenced peptides from rabbit eEF-2 were 100% identical to C.elegans eEF-2 kinase, then the two peptides should also match thesequence of mouse eEF-2 kinase. Degenerate primers were designed basedon the amino acid sequence of the peptides and were used to performRT-PCR on mouse spleen poly(A)⁺ mRNA. A single PCR product of ˜1.6 kbwas obtained and sequenced. To obtain the full-length cDNA, 5′ RACE and3′ RACE were performed using mouse spleen cDNA. The full-length cDNA,which encodes 724 amino acids, was expressed in a cell-free coupledtranscription/translation system. A single translation product with anapparent molecular weight of 100 kDa was obtained.

A cDNA for rat eEF-2 kinase was cloned and sequenced using a fragment ofmouse eEF-2 kinase cDNA to probe a PC12 cDNA library. However, afterthis work was completed, a paper describing the cloning of eEF-2 fromrat skeletal muscle was published (Redpath et al., (1996) J Biol. Chem.271:17547–17554) and the reported sequence appears to be identical tothe eEF-2 kinase sequence from PC12 cells. Like the mouse eEF-2 kinase,the rat eEF-2 kinase cDNA encodes a 724-amino acid protein.

The human eEF-2 kinase cDNA was cloned. RT-PCR was performed on poly(A)⁺mRNA from the human glioma cell line T98G using 20′mer primerscorresponding to the 5′ and 3′ ends of the mouse eEF-2 kinase codingregion. The human eEF-2 kinase cDNA encodes a 725 amino acid protein.

EXAMPLE 2 Lack of Homology of eEF-2 Kinase to Members of EukaryoticProtein Kinase Superfamily

The alignment of the amino acid sequences of C. elegans and mammalianeEF-2 kinases is shown in FIG. 2. Rat and mouse eEF-2 kinase are verysimilar being 97% identical and differing by only 23 amino acids. HumaneEF-2 kinase is 90% identical to mouse and rat eEF-2 kinase. Incontrast, C. elegans eEF-2 kinase is found to be only 40% identical tomammalian eEF-2 kinase.

According to the current classification, eEF-2 kinase belongs to thefamily of closely related calmodulin-dependent protein kinases.Surprisingly, upon analyzing eEF-2 kinase sequences, we did not find anyhomology to the other calmodulin-dependent kinases or to any othermembers of the protein kinase super-family. The only motif which itshares with all other protein kinases is the GXGXXG (SEQ ID NO: 21)motif (279–284 in C. elegans eEF-2 kinases; 295–300 in mouse eEF-2kinase) which forms a glycine-rich loop and is part of the ATP-bindingsite. Comparison of mammalian and C. elegans eEF-2 kinase revealed onlyone extended region of homology that spans ˜200 amino acids upstream ofthe GXGXXG motif. The high degree of similarity and the proximity to thenucleotide-binding site suggests that these 200 amino acids representthe catalytic domain. This region has a high degree of similarity and aportion of this region (amino acids 251–300 in mouse eEF-2 kinase)displays 75% identity to the catalytic domain of MHCKA (see below),which also suggests that this is the catalytic domain. In the recentlypublished rat eEF-2 kinase sequence [Redpath et al., J. Biol. Chem. 271:17547–17554 (1996)], the catalytic domain was predicted to residebetween amino acids 288 and 554 based on the homology with the catalyticdomain of cAMP-dependant protein kinase (PKA). Our results demonstratethat their prediction cannot be correct for several reasons. First, wefind that the homology of this region with PKA is not statisticallysignificant. Second, this region is the least conserved betweenmammalian and C. elegans eEF-2 kinase. Finally, according to secondarystructure predictions [made by Alexei V. Finkelstein, Institute ofProtein Research, Russia using the ALB-GLOBULE program [Ptitsyn andFinkelstein, Biopolymers 22:15–25 (1983)]], this region most likely hasa distorted structure and contains almost no α:-helices or β-strands,which are characteristic of a catalytic domain.

Because eEF-2 kinase is CA²⁺/calmodulin-dependant, it should contain acalmodulin-binding domain, which is usually represented by anamphipathic α-helix. There are several regions that could possiblyassume an amphipathic α-helical conformation. Further biochemicalanalysis is required to determine which of these is thecalmodulin-binding domain.

In the C-terminal region, there is a short stretch of 22 amino acidswhich is 86% identical between mammalian and C. elegans eEF-2 kinase andis preceded by a longer region of weak homology. We do not know thefunction of this conserved region at present. One of the possibilitiesis that it is that it is involved in oligomerization of the kinase. Itwas thought previously that eEF-2 kinase was an elongated monomerbecause it migrated during gel filtration as an ˜150-kDa protein andmigrated on SDS gels as a 105-kDa polypeptide [Ryazanov and Spirin,Translational Regulation of Gene Expression, Pienum, N.Y., Vol 2, pp433–455 (1993); Abdelnajid et al., Int. J. Dev. Biol., 37:279–290(1993)]. However, the molecular weight of a monomer of mammalian eEF-2kinase based on the predicted sequence is just 82 kDa. Thus, it ispossible that eEF-2 kinase is not a monomer but a responsible fordimerization. Interestingly, according to computer prediction using theCOIL program, this conserved region can form a coiled-coil. Formation ofcoiled-coil is often responsible for dimerization [Lupas, TrendsBiochem. Sci., 21:375–382 (1996)].

We found that eEF-2 kinases is homologous to the central portion of therecently described MHCKA from Dictyostelium [Futey et al., J. Biol.Chem. 270:523–529 (1995) see FIG. 2]. The kinase was biochemicallyidentified as a 130-kDa protein and has a demonstrated role in myosinassembly, both in vitro and in vivo [Futey et al., 1995, supra]. As witheEF-2 kinase, MHCKA displays no region with detectable similarity to theconserved catalytic domains found in known eukaryotic protein kinases.Primary structure analysis of MHCKA revealed an amino-terminal domainwith a probable coiled-coil structure, a central nonrepetitive domain,and a C-terminal domain consisting of seven WD repeats [Futey et al.,1995, supra]. A fragment of the central nonrepetitive domain of MHCKAcontaining amino acids 552–841 was recently shown to represent thecatalytic domain [Cote et al., J. Biol. Chem. 272:6846–6849 (1997)].

Because the catalytic domain of MHCKA and eEF-2 kinase have a highdegree of similarity, the substrate specificity of these two kinases wasassayed. It was demonstrated that MHCK A cannot phosphorylate eEF-2, andlikewise, rabbit eEF-2 kinase cannot use myosin heavy chains as asubstrate. This demonstrated that each of these kinases is specific fortheir respective substrates.

EXAMPLE 3 eEF-2 Kinase and MHCKA Define A New Class of Protein Kinases

Members of the eukaryotic protein kinase superfamily are characterizedby a conserved catalytic domain containing approximately 260 amino acidsand is divided into twelve subdomains [Hanks and Hunter, FASEB J.,9:576–596 (1996); Hardie and Hanks, The Protein Kinase Facts Book,Academic, London (1995), Taylor et al., Annu. Rev. Cell Biol. 8:429–462(1992) Johnson et al., Cell. 85: 149–158 (1996)]. The three-dimensionalstructure of several protein kinases revealed that the catalytic domainconsists of two lobes. The smaller N-terminal lobe, which has a twistedβ-sheet structure, represents the ATP-binding domain. The largerC-terminal lobe, which is predominantly α-helical is involved insubstrate binding. At the primary structure level, the only motifsimilar between eEF-2 kinase, MHCK A, and other protein kinases is theGXGXXG motif which forms the loop interacting directly with thephosphates of ATP [Hanks and Hunter, 1996, supra; Hardie and Hanks 1995,supra; Taylor et al., supra]. In eukaryotic protein kinases, this motifis located at the very N terminus of the ATP-binding lobe of thecatalytic domain. In contrast, in a eEF-2 kinase and MHCK A, this motifis close to the C terminus of the catalytic domain (see FIG. 3).However, the overall topology of the ATP-binding subdomain of eEF-2kinase and MHCK A can be similar to other protein kinases because theregion upstream of the GXGXXG (SEQ ID NO: 21) motif is stronglypredicted to contain four or five β-strands and thus can form a twistedβ-sheet.

However, the mechanism of ATP-binding to eEF-2 kinase is probably quitedifferent in comparison to other conventional members of the eukaryoticprotein kinase superfamily. In protein kinases, there is a conservedlysine residue, corresponding to Lys-72 in cAMP-dependant proteinkinases which binds to the β- and γ-phosphates of ATP and is located atabout 20 amino acids downstream of the GXGXXG motif. Analysis of eEF-2kinase and MHCK A sequences revealed that there are no conserved lysineresidues in the vicinity of the GXGXXG motif. There is another atypicalprotein kinase, BCR-ABLE, which does not contain this conserved lysineand it is proposed that it interacts with ATP via two cysteine residues[Maro and Witte, Cell, 67:459–468 (1991)]. Interestingly, eEF-2 kinaseand MHCK-A contain two conserved cysteine residues (Cys-313 and Cys-317in mouse eEF-2 kinase) which are located near the GXGXXG motif andtherefore might be involved in ATP binding. Thus the mechanism ofATP-binding of eEF-2 kinase and MHCK A is different from other membersof the protein kinase superfamily, but may be similar to that of theBCR-ABLE protein kinase.

The overall catalytic mechanism of eEF-2 kinase and MHCKA is probablyalso very different from other eukaryotic protein kinases. All membersof the eukaryotic protein kinase superfamily contain a DXXXN (SEQ ID NO:22) motif in the catalytic loop and a DFG motif in the activationsegment [Hanks and Hunter, 1996; supra, Hardie and Hanks 1995, supra;Taylor et al., supra; Johnson et al., 1996, supra]. These two motifs,which are directly involved in the catalysis of the proteinphosphorylation reaction, are absent from the eEF-2 kinase and MHCK Acatalytic domain.

We would predict that there are other protein kinases which arestructurally similar to eEF-2 kinase and MHCK A. An extensive search ofthe entire nonrestricted database of the National Center forBiotechnology Information using the BLAST program did not reveal anyprotein with a significant homology to the catalytic domain of eEF-2kinase and MHCKA. A search of the Expressed Sequence Tag (EST) databaserevealed several ESTs from C. elegans, mouse and human which areessentially identical to portions of eEF-2 kinase cDNA sequencesreported here. Interestingly, a search of the recently completed genomedatabase of Saccharomyces cerevisiae did not reveal any protein withhomology to eEF-2 kinase despite the fact that eEF-2 phosphorylation wasreported in yeast (41).

Conclusion.

Since the catalytic domains of eEF-2 kinase and MHCK A do not sharehomology with other known protein kinases, these two protein kinasesestablish the presence of a novel and widespread superfamily ofeukaryotic protein kinases. Although the existence of several unusualprotein kinases have been reported, to our knowledge, we demonstrate forthe first time the existence of a biochemically well-characterized andubiquitous protein kinase that is structurally unrelated to otherserine/threonine/tyrosine kinases. Contrary to the widely acceptedbelief that all eukaryotic protein kinases evolved from a singleancestor, our results suggest that eukaryotic protein kinases appearedat least twice during the course of evolution. This also suggests that,in addition to the relatively well-characterized catalytic mechanismemployed by members of eukaryotic serine/threonine/tyrosine proteinkinase superfamily, there exists another mechanism of protein kinasesuperfamily, there exists another mechanism of protein phosphorylation.Further studies will reveal the molecular details of this mechanism andwhether there are other protein kinases that phosphorylate theirsubstrates using this mechanism.

EXAMPLE 4 Cloning and Analysis of Melanoma Alpha Kinase cDNA

Here, we describe cloning and sequencing of a novel protein entitled“melanoma alpha-kinase”. This protein has two domains, one domain is thealpha-kinase catalytic domain and the other is an ion channel. This isthe first example of an ion channel being covalently linked to a proteinkinase. It is likely that this novel protein kinase can be regulated byion flow through the membrane. Expression of this kinase was detected inall mouse tissues studied, including heart, skeletal muscle, brain,liver and lung. This kinase is the most abundant in the heart. The ionchannel portion is very similar to (70% identical) to a previouslyidentified protein called melastatin that is selectively downregulatedin metastatic tumors, and therefore is believed to be a metastasissuppressor gene. Melanoma alpha-kinase, as well as melastatin, belongsto the TRP family of ion channels. All TRP proteins function astetramers, and various trp proteins can form tetramers in differentcombinations that results in ion channel with different properties.Considering the high degree of similarity between melanoma kinase andmelastatin, it is likely that melanoma kinase can form tetramericcomplexes with melastatin. In humans, melanoma kinase is located onchromosome 15q21.

Human EF-2 kinase amino acid sequence (Acc. No. AAB58270) was used tosearch for homologous sequences in the expressed sequence tag (EST)division of Genbank using the BLAST server and the tblastn program atthe National Center for Biotechnology Information. One human EST (Acc.No. AA332887) that overlapped and displayed significant homology to thecatalytic domain of EF-2 kinase was found. We used the nucleotidesequence of this EST to look for overlapping EST sequences. Weidentified mouse melanoma EST sequence (Acc. No. AA138771) thatoverlapped by 45 nucleotides with the human EST sequence. We obtainedthe mouse melanoma EST clone from Research Genetics and sequenced theentire clone. This clone represents the 3′ end of melanoma alpha-kinasemRNA and includes the 3′ untranslated region plus approximately 350amino acids of the C-terminus of the protein.

To obtain the full-length cDNA for mouse melanoma alpha-kinase, we useda Marathon-ready mouse heart cDNA library from Clontech. To obtain theremaining sequence of melanoma alpha-kinase, we performed 5′ rapidamplification of cDNA ends (RACE) using the following primers: MK1-R1(5′-TGACCAGGTACACAGCACTTTGACTGCTCT-3′ (SEQ ID NO: 23)). PCR wasperformed under the following conditions: denaturation for 15seconds at95° C.; annealing plus extension for 4 minutes at 68° C., 30 cycles. Asingle PCR product of approximately 4.0 kb was obtained and gel-purifiedusing a gel extraction kit (Qiagen). The fragment was ligated intovector pCR2.1 using the TA cloning kit (Invitrogen), and thentransformed into Escherichia coli TOP10F′. Plasmid DNA was purified, andrestriction analysis used to verify the orientation of the codingsequence with respect to the T7 promoter. Three clones were chosen andsequenced using an ABI 377 sequencer (Applied BioSystems).

To obtain full-length human melanoma alpha-kinase cDNA, new primers weredesigned for 5′ and 3′ RACE using sequence information from mousemelanoma alpha kinase cDNA fragments, and full-length cDNA was obtainedusing a human leukocyte cDNA library (provided by Dr. S. Kotenko).

Mouse melanoma alpha kinase hybridizations were performed using EST585207 DNA as a probe. The probe was labeled with [a-32P]dCTP using therandom-primed DNA labeling method. A multiple tissue northern blot(CLONTECH) was prehybridized at 42° C. for 16 hours in a 50% formamidesolution containing 10× Denhardt's solution, 5×SSPE, 2% SDS, and 100μg/ml salmon sperm DNA. Hybridizations were completed in the samesolution containing the 32P-labeled probe (1×106 cpm/ml; specificactivity, 1×108 dpm/μg DNA) and 10% dextran sulfate at 42° C. for 16hours. Blots were washed twice at room temperature (15 minutes) in2×SSPE, 0.05% SDS, and once at 50° C. (15 minutes) in 0.5×SSPE, 0.5%SDS. RNA/cDNA hybrids were visualized by autoradiography.

EXAMPLE 5 A Novel Type of Signaling Molecule-Protein Kinases CovalentlyLinked to Ion Channels

Abstract

Recently we identified a new class of protein kinases with a novel typeof catalytic domain structurally and evolutionarily unrelated to theconventional eukaryotic protein kinases. This new class, which we namedalpha-kinases, is represented by eukaryotic elongation factor-2 kinaseand the Dictyostelium myosin heavy chain kinases. Here we cloned andsequenced five other mammalian alpha-kinases. One of these proteins,which was initially identified as an EST from a mouse melanoma cDNAlibrary, was named melanoma alpha-kinase (MK), and according to northernanalysis, has a ubiquitous tissue distribution, being present in allmouse and human tissues studied. Four other alpha kinases have a morerestricted tissue distribution and were named after the tissue in whichthey are predominantly expressed: kidney alpha-kinase (KK), heartalpha-kinase (HK), skeletal muscle alpha-kinase (SK), and lymphocytealpha-kinase (LK). All these protein kinases are large proteins of morethan 1000 amino acids with a typical alpha-kinase catalytic domainlocated at the very carboxyl-terminus. We expressed the catalytic domainof human MK in Escherichia coli, and found that it autophosphorylates onthreonine residues, demonstrating that it is a genuine protein kinase.

Unexpectedly, we found that the long amino-terminal portions of melanomaand kidney α-kinases represent new members of the transient receptorpotential (TRP) ion channel family, which are implicated in themediation of capacitative Ca²⁺ entry in non-excitable mammalian cells.This suggests that melanoma and kidney α-kinases, which represent anovel type of signaling molecule, are involved in the regulation of Ca²⁺influx into mammalian cells. It has also been implied that TRP channelsmay mediate the Ca2+-release-activated Ca2+ current (CRAC). The channelportions of KK and MK were highly similar to each other and highlysimilar to melastatin. Melastatin is a putative Ca2+ channel that wasidentified as a gene product specifically downregulated in metastaticmelanoma. Phylogenetic analysis revealed that both KK and MK belong tothe long TRP (LTRP) channel subfamily, which also includes melastatin,and several uncharacterized channel proteins from mammals,Caenorhabditis elegans and Drosophila. Among LTRP channels, only MK andKK possess an α-kinase domain.

Introduction

The vast majority of eukaryotic protein kinases have a typical catalyticdomain structure consisting of twelve conserved subdomains (1). Theexistence of other protein kinases with a different structure wasreported in eukaryotes (2–4). Recently we identified a new class ofprotein kinases with a novel type of catalytic domain, structurally andevolutionarily unrelated to the conventional eukaryotic protein kinases(5). This class, which we named alpha-kinases, is represented byeukaryotic elongation factor-2 (eEF-2) kinase and the Dictyosteliummyosin heavy chain kinases A and B (MHCKA and B) (2,3,29). The catalyticdomain of the alpha-kinases can be subdivided into eight domains (6).There is no significant homology between those eight domain and any ofthe twelve subdomains of the conventional protein kinases. We named thisnew class of protein kinases the alpha-kinases because the existingevidence suggests that they can phosphorylate amino acids located withina-helices (6).

In order to study how widespread the alpha-kinases are and to identifynew members of the alpha-kinase family in mammals, we used a functionalgenomic approach. We performed an extensive expressed sequence tag (EST)database search for sequences homologous to the catalytic domain ofhuman eEF-2 kinase. As a result of this screen, we obtained severalpartial sequences for putative alpha-kinases, which were subsequentlyused to clone the full-length cDNAs.

We cloned and sequenced and analyzed the tissue distribution of five newmembers of the alpha-kinase family. All these proteins contain thetypical alpha-kinase catalytic domain. The expressed catalytic domain ofMK was able to autophosphorylate, therefore demonstrating that it is agenuine protein kinase.

Unexpectedly, the amino-terminal portions of MK and KK appear to have along amino-terminal portions highly homologous to a number of ionchannel proteins that belong to transient receptor potential (TRP)family of Ca2+ channels. The ion channel portions of MK and KK areremarkably homologous to melastatin. Melastatin is a proteindownregulated in metastatic melanoma, and is a newly discovered memberof the TRP Ca2+ channel family (7, 8). TRP channels derive their namefrom a mutation in a Drosophila calcium channel that is involved inphotoreception (30,31). This mutation caused an inability to maintain asustained receptor potential, and was therefore named transient receptorpotential (trp), and the channel was named the TRP channel (19,30,31).Several homologues of TRP channels in mammals have been identified(27,32,33). The recent interest in the TRP channel family is related tothe fact that they may represent channels responsible for store-operatedcalcium influx—one of the major pathways of calcium entry intonon-excitable mammalian cells (9,10,12,18,24,34).

TRP channels are Ca2+-permeable channels believed to be responsible forCa2+ influx in response to depletion of internal Ca2+ stores (9–11). Theion channel portion of MK and KK, being highly similar to melastatin,makes MK and KK new member of the TRP family, in particular the LTRPfamily to which melastatin belongs.

Thus, in our work, we demonstrated a novel type of signaling molecule—anion channel covalently linked to a protein kinase. We discuss thepossibility that this hybrid ion channel/protein kinase is involved inthe regulation of store-operated Ca2+ entry in non-excitable tissues.

Materials and Methods

Cloning of Melanoma Kinase

We searched the EST database, and identified several mouse and humanESTs homologous to the catalytic domain of human eEF-2 kinase. One ofthese EST clones was derived from a mouse melanoma cDNA library (IMAGEclone 585207; GenBank accession #AA138771). We sequenced this clone andfound it encodes the C-terminus (approximately 300 amino acids) of anovel protein. We used 5′ rapid amplification of cDNA ends (RACE) and amouse heart Marathon-ready cDNA library (Clontech) to determine thefull-length sequence. We used this sequence information to designprimers and clone human melanoma kinase from a Hela cell cDNA library.

Cloning of Kidney Kinase

A further search in the EST database revealed an EST derived from amouse kidney cDNA library encoding another protein homologous to thecatalytic domain of eEF-2 kinase (IMAGE clone 656119; GenBank accession# AI390333). We used the sequence information to perform 5′RACE and3′RACE using a mouse heart Marathon-ready cDNA library (Clontech). Afterpartial sequencing of this clone, we used the new sequence informationto design primers and clone human kidney kinase from a human kidneyMarathon-ready cDNA library (Clontech).

Cloning of Heart Kinase

Database searches revealed another homologous EST clone approximately 2kb in length (IMAGE clone #585879; GenBank accession #AA140393). Toobtain the full length cDNA, we screened a mouse heart 5′-STRETCH PLUScDNA lambda library (Clontech). A 32P-labeled 2 kb EST fragment was usedas a probe for clone identification. Several clones gave positivesignals, and were further analyzed by PCR. The largest of the clones (˜5kb) was sequenced. Subsequently we found a human EST in the EST databasehomologous to mouse heart kinase (IMAGE clone #843057; GenBank accession#AA485987). We sequenced this clone, and found it encodes a proteincorresponding to the C-terminus of heart kinase. After partialsequencing of this clone, we used the new sequence information to designprimers and clone human heart kinase from a human heart Marathon-readycDNA library (Clontech).

Cloning of Skeletal Muscle Kinase

We searched the HTGS database for sequences homologous to mouse heartkinase, and found a clone containing the gene encoding a protein similarto heart kinase. We designed primers using the database sequence andperformed PCR using a human placenta cDNA library to clone the catalyticdomain of muscle kinase.

Cloning of Lymphocyte Kinase

By searching the non-restricted database, we found a genomic DNA clonederived from chromosome IV that encodes a protein containing theα-kinase catalytic domain. We used this sequence information to searchfor overlapping ESTs in order to reconstruct the full-length protein,and then to design primers for PCR to clone the full-length protein froma lymphocyte cDNA library.

Cloning of the Melanoma Kinase Catalytic Domain

The catalytic domain of melanoma kinase was cloned from a Hela cell cDNAlibrary. Primers were designed based upon the sequence of melanomakinase. The sequences of the primers are:

Forward: 5′-GTTAGTACACCATCTCAGCCAAGTTGCAAA-3′, (SEQ ID NO: 24) Reverse:5′-TTATAACATCAGACGAACAGAATTAGTTGATTCTGATTCT-3′ (SEQ ID NO: 25).PCR conditions were as follows: 30 sec. at 94° C., 30 sec. at 58° C., 3min. at 72° C. for 30 cycles, followed by a 10 min. final extension at72° C. The PCR product was cloned into PCRII-TOPO vector (Invitrogen) asper manufacturer's instructions. The insert was then subcloned into theEcoRI site in pMAL-p2x (NEB) to tag the protein with maltose bindingprotein (MBP).Expression and Purification of MBP-MK

E. coli strain DH5α carrying the pMALp2x-MK plasmid were grown to anOD1=600 0.5, then IPTG was added to a final concentration 0.3 mM. Cellswere grown for additional 6 hours at 37° C. All following procedureswere carried out at 4° C. Cells was resuspended in 20 mM Tris-HCl (pH7.4), 200 mM NaCl, 1 mM EDTA, 10 mM b-mercaptoethanol and sonicated.Inclusion bodies were pelleted by centrifugation at 30,000× g for 30min., dissolved in 6M urea, 20 mM Tris-HCl (pH 7.4), 200 mM NaCl, 1 mMEDTA, 10 mM b-mercaptoethanol, 20% (w/v) glycerol and centrifuged againat 30,000×g for 30 min. The supernatant was dialyzed overnight againstthe same buffer but without urea. After dialysis, the sample wascentrifuged once again at 30,000×g for 30 min., and the supernatant wasloaded onto an amylose column equilibrated with 20 mM Tris-HCl (pH 7.4),200 mM NaCl, 1 mM EDTA, 10 mM b-mercaptoethanol, 20% (w/v) glycerol.Elution was performed by a step gradient of the same buffer plus 10 mMmaltose.

Phosphorylation of MBP-MK

Assays for MBP-MK activity were performed at 30° C. in an assay buffercontaining 50 mM Hepes-KOH (pH 6.6), 10 mM MgCl2, 1 mM DTT, 50 mM ATP,20 mCi [g-32P]-ATP and 3mg MBP-MK. After incubation, Laemmli samplebuffer was added, samples were boiled and 25 ml of each sample wasloaded onto a 10% SDS-PAGE gel. The gel was stained with Coomassie Blue,dried and exposed to film for 16 hours.

Phosphoamino Acid Analysis

Phosphorylation of MBP-MK was done as described above, but the reactionvolume was increased 5-fold. The incubation time was 2 hours. Sampleswere separated by 10% SDS-PAGE and transferred to an Immobilon-Pmembrane (Millipore). The portion of membrane with phospho-MBP-MK wasexcised and incubated in 6M HCl at 110° C. for 1 hour. After incubation,the mixture was dried, and the dried pellet was dissolved in 9 μl ofwater, with the addition of non-radioactive phosphoserine,phosphothreonine, and phosphotyrosine. Phosphoamino acids were separatedby thin-layer chromatography on cellulose (cellulose on polyester;Aldrich) using a buffer consisting of isobutyric acid and 0.5M NH₄OH ina 5:3 ratio. The TLC plate was stained with 0.2% ninhydrin and exposedto film.

Northern Blot Analysis

Standard Multiple Tissue Northern (MTN) Blots (Clontech) were stainedwith DNA probes according to manufacturer's instructions. The probeswere labeled with [α-³²P]-dCTP using the Megaprime DNA labeling system(Amersham) using specific DNA fragments as templates. The DNA fragmentswere obtained as follows.

A 430 bp DNA fragment of human melanoma kinase (corresponding tonucleotides 4331–4761) was obtained by PCR from a Hela cDNA library(Clontech). The following primers were used for PCR:

LMK2: 5′CTGCGACAGAGACTACATGGGGTAGAACTC 3′ (SEQ ID NO: 42) LMK4:5′TGAGTGTCTTCGGTAGATGGCCTTCTACTG 3′ (SEQ ID NO: 43)

A 741 bp DNA fragment of human kidney kinase was produced by PCR from aplasmid containing kidney kinase cDNA. The region corresponding tonucleotides 3721–4462 was amplified using the following primers:

KK-F1: 5′ATGGAGATTGCTGGAGAGAAG 3′ (SEQ ID NO: 44) and KK-R3:5′ATTCACTACTCTGGGCCGATC 3′ (SEQ ID NO: 45)

A 1.2 kb DNA fragment of human muscle kinase was obtained by EcoRIdigestion of the human muscle kinase cDNA insert cloned in pCRII-TOPO(Invitrogen). The mouse melanoma kinase 2.2 kb DNA fragment was obtainedfrom EST clone 585207. The fragment was cut out with BamHI and XhoI frompBluescript SK-vector (Stratagene). The mouse heart kinase 2 kb DNAfragment was produced by restriction with SmaI and KpnI from EST clone585879.

Results

In our work we cloned and sequenced five new members of the alpha-kinasefamily. They are named according to their tissue distribution: heartalpha-kinase (HK), melanoma alpha-kinase (MK), kidney alpha-kinase (KK),skeletal muscle alpha-kinase (SK), lymphocyte alpha-kinase (LK). Thenucleic acid sequence and predicted amino acid sequence of the humanheart alpha-kinase (HK) are depicted in FIG. 8A and B (SEQ ID NO: 34 and35, respectively). The nucleic acid sequence and predicted amino acidsequence of the mouse heart alpha-kinase (HK) are provided in SEQ ID NO:36 and 37, respectively. The nucleic acid sequence (SEQ ID NO: 26) andpredicted amino acid sequence (SEQ ID NO: 27) of the human melanomaalpha-kinase (MK) are depicted in FIG. 7A and B. The nucleic acidsequence (SEQ ID NO: 28) of mouse melanoma α-kinase is shown in FIG. 5.The predicted amino acid sequence (SEQ ID NO: 29) of mouse melanomaα-kinase is shown in FIG. 6. The nucleic acid and predicted amino andsequence of the human kidney alpha-kinase (KK) are depicted in FIG. 9Aand B (SEQ ID NO: 30 and 31, respectively). The nucleic acid andpredicted amino and sequence of mouse kidney alpha-kinase (KK) areprovided in SEQ ID NO: 32 and 33, respectively. FIG. 10A and B depictsthe nucleic acid sequence (SEQ ID NO: 38) and predicted amino acidsequence (SEQ ID NO: 39) of human skeletal muscle alpha-kinase (SK). Thenucleic acid sequence (SEQ ID NO: 40) and predicted amino acid sequence(SEQ ID NO: 41) of the lymphocyte alpha-kinase (LK) are shown in FIG.11A and B. All of these protein kinases are large proteins of more than1000 amino acids with a typical alpha-kinase catalytic domain located atthe very C-terminus. FIG. 12 shows the alignment of the catalyticdomains of the cloned alpha-kinases. The catalytic domain sequencereveals 30–80% similarity between these alpha-kinases. It can be dividedinto several subdomains with no homology between these subdomains andany of the twelve subdomains of the conventional protein kinases.Altogether, there are sixteen positions in the alignment of the clonedproteins that are invariant among all the known alpha-kinases. All fivenew proteins are homologous to each other, as well as homologous to theeEF-2 kinase and MHCK B catalytic domains. A comparison of the five newα-kinases, eEF-2 kinase and MHCK B reveals sixteen invariant aminoacids. We reported previously (6) that the α-kinase catalytic domain canbe divided into eight subdomains, each having a characteristic sequencemotif. Identification of new α-kinases allowed us to characterize thesesubdomains more precisely. In addition, we used the ALB secondarystructure prediction service(http://indy.ipr.serpukhov.su/˜rykunov/alb/) (35) to predict a consensussecondary structure of the α-kinase catalytic domain. Subdomain I beginswith a conserved Trp residue present in all α-kinases with the exceptionof HK, which has a Phe in this position. Subdomain I is alsocharacterized by an invariant Gly and an invariant Arg that are part ofthe conserved Arg-Lys-Ala motif. Subdomain II is characterized by aninvariant Lys, that is part of an Hyd-Hyd-X-Lys motif (Hyd=hydrophobic).Subdomain III contains an invariant Gln, and is predicted to form anα-helix. Subdomain IV contains a stretch of hydrophobic amino acids andis predicted to form a β-sheet. Subdomain V is predicted to form anα-helix containing an invariant Glu, and is followed by a conserved Asn(an Arg in HK and SK). Subdomain VI is predicted to form a β-turn-β-turnstructure with an invariant His in the first P-strand, and the conservedsequence, Leu-Leu-Val-Val-Asp-Leu-Gln-Gly, that forms the end of thesecond β-strand and second turn and also contains an invariant Asp andGly. Subdomain VII is characterized by the conserved sequence,Leu-Thr-Asp-Pro-Gln-Ile, which contains an invariant Thr-Asp. SubdomainVIII begins with a Gly-rich region that is not predicted to have anyregular secondary structure, followed by a sequence containing invariantPhe and His residues, an invariant Cys-Asn-X-X-Cys motif and aninvariant Leu. The region containing the Cys-Asn-X-X-Cys motif ispredicted to form a short α-helix.

Phylogenetic analysis (FIG. 13) of the cloned alpha-kinases suggeststhat all five alpha-kinases are more closely related to each other thanto eEF-2 kinase or the MHCKs, and form a separate subfamily. MK isclosely related to KK (78% identity). HK is similar to SK (47%identity). LK has less similarity to the others, but they all form adistinctive separate subfamily of alpha-kinases, displaying variousdegrees of similarity to eEF-2 kinase.

In order to analyze kinase activity, we expressed the catalytic domainof MK as maltose-binding protein (MBP) fusion protein. Affinity-purifiedMBP-MK was able to autophosphorylate. FIG. 14 shows the time course of32P incorporation into MBP-MK. This phosphorylation can be reversed byincubation with lambda phosphatase. Phosphoamino acid analysis revealedthat MK is phosphorylated on a threonine residue.

Using northern blot analysis, we analyzed the tissue distribution of theα-kinases in human and mouse tissues (FIG. 15). MK, KK, HK and SK arelarge proteins (corresponding cDNAs are 7.5kb). LK mRNA is representedby two bands, 5.5 and 7.5 kb. An additional minor band at 9.5 kb can beseen for SK. MK is ubiquitously expressed, being detected in everytissue tested. In human tissues, it is most abundant in the liver,kidney and heart, and in mouse tissues—heart, lung, liver and kidney.There were noticeable amounts in human lymphoid, bone marrow and thymustissues. The least amount of MK mRNA, among human tissues, was observedin brain. LK mRNA can be detected by northern analysis in various humantissues. Its tissue distribution was virtually identical to MK, althoughit is much less abundant than MK since three weeks of exposure wererequired to visualize the bands. LK can be detected by reversetranscriptase-PCR (RT-PCR) in human fetal liver and placenta tissues,and lymphocyte libraries. A full-length clone was obtained from alymphocyte cDNA library. KK is present almost exclusively in kidneytissue, with trace amounts in human lymphocyte, brain and bone marrowtissues. HK is found almost exclusively in mouse heart tissue, with asmaller amount in skeletal muscle tissue. It was not seen in any othertissues tested. SK is very abundant in human muscle tissues, with aconsiderable amount in human heart tissue. Trace amounts of SK can beseen in human lung, placenta and kidney tissues.

We cloned full-length cDNA for MK and KK. The long N-terminal portion ofMK and KK display high similarity to ion channels homologous to TRPchannel family. The MK sequence contained 1864 amino acids and the KKsequence contained 2011 amino acids. Unexpectedly, we found that thelong amino terminal portions of MK and KK are homologous to ionchannels. The approximately 1200 amino acid long N-terminal portions ofMK and KK were similar to each other (59% identical) and homologous tomelastatin (48% and 51% identical respectively; see FIG. 17). Melastatinis a putative Ca²⁺ channel that belongs to the transient receptorpotential (TRP) family of ion channels (8). Like melastatin, MK and KKcontain all the sequence elements characteristic of the TRP channelfamily. These elements include six predicted transmembrane segments, ahighly conserved sequence in the putative pore region betweentransmembrane segments 5 and 6, a highly conserved sequence at the endof transmembrane segment 6, and a Pro-Pro-Pro motif-containing sequencethat immediately follows transmembrane segment 6 (FIG. 17). We foundseveral human, mouse, Caenorhabditis elegans and Drosophila proteins inGenBank that are highly similar to the melastatin-like portions of MKand KK (FIG. 17). All these proteins belong to a subfamily of the TRPchannels which were named long TRP channels (LTRPC), and arecharacterized by a long conserved N-terminal sequence that precedes thetransmembrane segments (14). These proteins include a protein that wasinitially called TRPC7 (36), and was later renamed LTRPC2 (14), aprotein named MTR1 or LTRPC5 (14,37), an unnamed human putative proteinwe named LTRPC6 (GenBank accession #AK000048), three C. elegans proteins(F54D1.5, T01H8.5, and C05C12.3) named respectively CeLTRPC1, CeLTRPC2and CeLTRPC3 (14), and a Drosophila putative protein that we namedDmLTRPC1 (GenBank accession #AE008311) (FIG. 17). Thus, MK and KK can beclassified as members of the LTRPC subfamily, and we suggest they bedesignated LTRPC3 and LTRPC4, respectively. As can be seen in FIG. 17,MK and KK, like other LTRPC proteins, contain a long N-terminal sequence(approximately 600–800 amino acids) that has several conserved andunique motifs. The ALB program predicted several long α-helices in thisregion in all LTRPC proteins.

Phylogenetic analysis revealed that MK, KK as well as other LTRPCproteins are related to the prototypic Drosophila TRP protein, but aremore similar to each other than to the prototypic TRP (FIG. 19).

We also determined the full-length sequence of LK cDNA that encodes aprotein containing 1242 amino acids. The TMPred program predicts fourtransmembrane segments located close to the N-terminus.

HK and SK cDNAs encode proteins of 1531 and 1215 amino acids,respectively. Comparison of the predicted amino acid sequences of HK andSK with other proteins in GenBank using the BLAST program revealed thatthere is an approximately 100 amino acid motif located just N-terminalto the catalytic domain that displays sequence similarity toimmunoglobulin-like domains of titin, myosin light chain kinase, andseveral other proteins. The same region in both kinases is identified asan immunoglobulin-like domain using the CD-Search program. The remainderof the HK amino acid sequence did not display any strong similarity toany known proteins. The N-terminal portion of SK displayed a weaksimilarity to collagen, which may be attributed to the high glycine andproline content.

Discussion

In our previous work, we discussed the unique structure of eEF-2 kinaseand the Dictyostelium MHCKs, and suggested that they represent a newclass of protein kinases, the α-kinases (5,6). In this work, we clonedand sequenced five new members of the α-kinase class. Thus, togetherwith eEF-2 kinase, there are at least six distinct α-kinases in mammals.These six α-kinases encompass all sequences from vertebrate sourcesdeposited in GenBank thus far with homology to the α-kinases. Withapproximately 90% of the human genome represented in GenBank to date, itis likely we cloned most, if not all, of the mammalian α-kinases.Interestingly, in the C. elegans genome there is only one α-kinase(eEF-2 kinase) while none have been identified in the Drosophila, yeastand plant genomes. However, the α-kinases appear to be widespread amongthe protozoa: sequences encoding proteins with a high similarity to theα-kinases are present in the genomes of Trypanosoma, Leishmania, andAmoeba.

We cloned and sequenced five new members of the alpha-kinase family. Allof these kinases have a typical alpha-kinase catalytic domain locatedthe very carboxyl-terminus. The alignment of the cloned alpha-kinasecatalytic domains reveals eight subdomains characteristic of thealpha-kinases which have no significant homology to the conventionaleukaryotic protein kinase catalytic domain. An alignment of the α-kinasecatalytic domain revealed several characteristic motifs. These motifsare different from those that characterize the eukaryotic Ser/Thr/Tyrprotein kinase superfamily, suggesting that the α-kinases andconventional eukaryotic protein kinases are structurally andevolutionarily unrelated. The expressed catalytic domain of MK is ableto efficiently autophosphorylate. Tissue distribution of the new clonedproteins reveals that among the cloned alpha-kinases, MK has a widetissue distribution, while the others (KK, HK, LK and SK) are specificfor particular tissues. SK, which is specific to human skeletal muscle,is remarkably abundant. Phylogenetic analysis suggests that our clonedalpha-kinases are closely related to each other, and are distantlyrelated to eEF-2 kinase and the MHCKs, forming a distinctive subfamilyof alpha-kinases. All of the five new alpha-kinases probably evolvedfrom a common ancestor during the evolutionary process. Therefore, thealpha-kinase family has been enlarged and now includes five new members.

Recently the structure of a novel type of protein kinase catalyticdomain has been determined represented by the bacterial histidinekinases, EnvZ and CheA (38,39). The structure of the catalytic domain ofthe histidine kinases appears to be completely different from that ofthe Ser/Thr/Tyr protein kinase superfamily but utilizes a fold similarto Hsp90, DNA gyrase B, and MutL (Bergerat fold; 40). There are proteinkinases that are highly similar to the bacterial histidine kinases, butphosphorylate their substrates on serine residues [for example, plantphytochromes (41) and animal pyruvate dehydrogenase kinase (42)] or ontyrosine residues [DivL (43)], suggesting that protein kinases with theBergerat fold can phosphorylate amino acids other than histidine. Is itpossible that the α-kinases also use a similar fold? We noticed that thedistribution of consensus secondary structure elements predicted for theα-kinases using the ALB program is similar to the distribution ofsecondary structure elements in EnvZ as determined by NMR. Moreover, theconserved asparagine and invariant aspartic acid residues located insubdomains V and VI, respectively, may correspond to the invariantasparagine and aspartic acid residues located in the N and G1 boxes,respectively, of the histidine kinases. These residues play a crucialrole in ATP binding (38,39,40). The glycine-rich region in subdomainVIII of the α-kinases may correspond to the G2 box of histidine kinases,which is a highly mobile region that forms part of the “lid” of the ATPbinding site (38,39,40). In addition, the histidine residuesphosphorylated by histidine kinases are located within α-helices(39,44,45), suggesting that the catalytic domain of these proteinkinases is adapted to recognize α-helices. Finally, the overall topologyof some α-kinases (MK, KK and LK) with several transmembrane segments inthe N-terminal region of the molecule and catalytic domain located atthe C-terminal region resemble the topology of many histidine kinases(46). All these facts raise the possibility that the α-kinases and thehistidine kinases may be evolutionarily related.

The expressed catalytic domain of MK is able to autophosphorylate,demonstrating that it is a genuine protein kinase. Phosphoamino acidanalysis revealed that MK autophosphorylates exclusively on a threonineresidue. Interestingly, the only two other α-kinases for which thesubstrates have been identified, eEF-2 kinase and MHCK A, bothphosphorylate their substrates on threonine residues. Therefore, it ispossible that the α-kinases in general are specific forphosphothreonine.

Northern analysis reveals that MK and LK have a wide tissue distributionsuggesting a general function for these kinases, while KK, HK and SK areexpressed primarily within specific tissues, suggesting they havetissue-specific functions. Interestingly, the tissue distributionpatterns of MK and LK were virtually identical, suggesting that thesetwo proteins are similarly regulated and may have similar functions.

Remarkably, two of the new members, MK and KK, appeared to have ionchannels at the very amino-terminus that are highly homologous to theTRP channel family. TRP channels are Ca2+-permeable channels that arebelieve to be responsible for Ca2+ influx in response to depletion ofinternal Ca2+ stores (9–11). Such a depletion of intracellular Ca2+stores followed by activation of the Ca2+ entry mechanism at the plasmamembrane is called capacitative Ca2+ entry (CCE).

CCE is loosely defined as an influx of Ca2+ from the extracellular spacefollowing inositol 1,4,5-triphosphate (IP3) or other Ca2+-mobilizingagent-induced depletion of internal Ca2+ from the ER and SR. CCE plays acentral role in many aspects of cell signaling, and is present in manytypes of cells. It is an essential component of the cellular response tomany hormones and growth factors. (12, 13). The TRP gene of Drosophila(19, 20) and its homologue, TRP-like (TRPL; 21)together withrecently-discovered mammalian homologues (22–25) were suggested toencode the CCE channels (26, 27). The family of known TRPs and theirhomologues are conserved from worms to humans. All show the same basicchannel subunit structure with six putative transmembrane helices, and arange of motifs in the amino- and carboxyl-terminal regions (14).

It was recently suggested that by their functional properties andstructure, the TRP channel family can be subdivided into threesubfamilies: short TRP (STRP), osmoTRP (OTRP) and long TRP (LTRP) (14).MK and KK display similarity to melastatin, which, by its structure andfunction, can be classified as an LTRP (14). Melastatin is a putativeCa2+ channel identified recently as a gene specifically downregulated inmetastatic melanoma (8). Sequence analysis of the ion channel region ofMK and KK reveals a typical structure of LTRPs. The function of STRPincluding Drosophila TRP and many mammalian homologs is to mediate Ca2+influx subsequent to activation of phospholipase C. The OTRP subfamilyis Ca2+-permeable channels involved in pain transduction, chemo-,mechano- and osmoregulation. The function of the LTRP subfamily channelsis not yet well characterized.

LTRP channels have longer coding sequences than STRP and OTRP,particularly at their amino-termini. All TRP channels share similarstructure: they have six transmembrane segments and a pore-forming loopbetween the fifth and sixth segments (reviewed in 24,9,14,47). The sameconserved sequences are present in MK and KK (FIG. 4). The Pro—Pro—Promotif that follows the sixth transmembrane segment in MK and KK ischaracteristic for STRP and LTRP channels. LTRPs do not have the ankyrinrepeats characteristic of the STRPs and OTRPs (14). Instead, they have along N-terminus with a unique and highly-conserved sequence. As can beseen in FIG. 17, the long N-terminal portion of MK and KK also has thishighly conserved sequence. This region in MK and KK is predicted to formseveral long α-helices. In addition to MK and KK, we identified eightother LTRP channels among the sequences deposited in GenBank: four inmammals, three in C. elegans, and one in Drosophila. The first LTRP tobe identified was melastatin, a putative Ca²⁺ channel whose mRNA isspecifically downregulated in metastatic melanoma (7,8). MK and KK areparticularly similar to melastatin (more than 48% and 51% identity)suggesting that MK and KK may be a product of a recent evolutionaryevent—a fusion between the α-kinase catalytic domain and amelastatin-like ion channel. Thus, considering the striking similarityof MK and KK to LTRP channels, we suggest that MK and KK are ionchannels with a unique molecular structure—ion channels covalentlylinked to a protein kinase. FIG. 18 and FIG. 19 show a schematicrepresentation of the major domains of MK, KK as well as otherα-kinases, a phylogenetic tree of LTRP channels, and a proposedstructural model of MK and KK. Thus, MK and KK, having ion channel partwith the typical structure of LTRPs, can be considered a new member ofthe LTRP family. To date, MK and KK are the only known channelscovalently linked to a protein kinase catalytic domain.

It is possible that the other three α-kinases may also have regionshomologous to ion channels. LK has four predicted transmembrane segmentsnear its N-terminus, although this region is not similar to the TRPchannels. We did not find sequences homologous to ion channels at theN-termini of HK and SK, however, it is likely that we did not obtain thevery N-terminal regions of these proteins. TRP channels areCa2+-permeable channels believed to be responsible for Ca2+ influx inresponse to depletion of internal Ca2+ stores (9–12,18,24,27,32,34). TheTRP gene of Drosophila (19,31) together with recently-discoveredmammalian homologues (27,32,33) were suggested to encode store-operatedCa²⁺ channels, also known as capacitative Ca²⁺ entry channels(9,10,12,18,24,27,31,32,34).

The first of the Ca²⁺ -permeable store-operated channels to becharacterized in detail were those mediating Ca²⁺ -release-activatedcurrent (I_(CRAC)) (16,48,51). CRAC channels are highly Ca²⁺ selective,low conductance channels that mediate Ca²⁺ entry in response todepletion of Ca²⁺ from intracellular stores in various non-excitablecells, and play a central role in activation of lymphocytes,degranulation of mast cells, and possibly mitogenic stimulation ofvarious cells (16,48,51,54,55). The molecular identity of CRAC channelshas not yet been determined. It has been suggested that members of theTRP channel family may underlie I_(CRAC) (9,10,12,18,24,27,32,34,55).However, none of the TRP channels studied to date have all theproperties of CRAC channels (14). Nevertheless, TRP proteins arecurrently the most likely candidates for CRAC channels, and it has beensuggested that the CRAC channels may be hidden in the LTRP family whosefunction is largely unknown (14).

It is possible that MK and KK are indeed the “hidden” members of theLTRP channel family mediating I_(CRAC). The tissue distribution of MK(which is ubiquitously expressed in all tissues tested, and predominantin non-excitable tissues such as liver and lymphocytes) is consistentwith this idea. Moreover, the protein kinase domain can be part of thesignaling mechanism that modulates channel function. There is evidencethat protein phosphorylation is involved in both the activation of thestore-operated Ca²⁺ channels as well as in the regulation of channelclosure (54,56–59).

The conserved location of the transmembrane domain and catalytic domainslinked together reveals a new structure for a novel type of protein.What is the role of such an unusual protein structure? There are anumber of reports indicating a role for protein phosphorylation in CCE.There are indications that protein phosphatases may regulate theresponsiveness of the entry channel to hypothetical diffusible componentof the entry, implying that the latter may act by promoting channelphosphorylation (34). It has been proposed that the endoplasmicreticulum might possess protein kinases or phosphatases capable ofaltering the phosphorylation state of the entry channel (12).Phosphatase inhibitors will enhance Ca2+ entry by serine/threoninephosphorylation (52). Tyrosine phosphorylation has been implicated incoupling store depletion of Ca2+ entry, but there are inconsistencies inoverall information and a possible explanation is that separate kinasesphosphorylate different components of the entry mechanism. Differentkinases may be involved in the process of CCE, one of the consistentimplications is that protein kinases possibly phosphorylate the IP3receptor, but the CRAC channel is the likely target for proteinphosphorylation (12). It was also shown that phosphatase inhibitors caninhibit ICRAC. The tissue distribution of MK is consistent with itsbeing a CRAC since it is present in all tissues and is most prominent innon-excitable, while barely detectable in the brain. We suggest that wediscovered a new type of molecule—a protein kinase covalently linked toan ion channel—represents a new signaling molecule which underlies CRACchannels. The placement of a kinase and channel on a single molecule isparticularly interesting and suggests a self-regulated molecule, wherebythe phosphorylation/autophosphorylation of these unique alpha kinasescontrols or contributes to the open or closed state of the channel. Inaddition, such an unusual molecular structure may be a part of a signaltransduction mechanism that links depletion of internal Ca2+ stores tochannel opening.

In summary, our discovery of five new members has broadened the class ofα-kinases. Two of the new α-kinases represent a novel type of signalingmolecule—a TRP-like ion channel covalently linked to a protein kinasesuggesting that one of the functions of the α-kinases is to regulateCa²⁺ influx in mammalian cells. It is also possible that MK and KK areCRAC channels and play a central role in the immune response.

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This invention may be embodied in other forms or carried out in otherways without departing from the spirit or essential characteristicsthereof. The present disclosure is therefore to be considered as in allaspects illustrate and not restrictive, the scope of the invention beingindicated by the appended claims, and all changes which come within themeaning and range of equivalency are intended to be embraced therein.

Various references are cited throughout this Specification, each ofwhich is incorporated herein by reference in its entirety.

1. An isolated nucleic acid encoding a mammalian alpha kinase expressedin the heart and having alpha kinase activity, wherein the nucleic acidis selected from the group consisting of: a. the DNA sequence of SEQ IDNO: 34; and b. DNA sequences capable of encoding the amino acid sequenceencoded by the DNA sequences of subpart (a).
 2. An isolated nucleic acidencoding human alpha kinase, expressed in the heart and having alphakinase activity, wherein the nucleic acid comprises the DNA sequence ofSEQ ID NO:
 34. 3. A recombinant DNA expression vector comprising thenucleic acid of claim 1, wherein the DNA encoding the alpha kinase isoperatively associated with an expression control sequence.
 4. Anisolated transformed host cell transfected with the DNA vector of claim3.
 5. A unicellular host transformed with a recombinant DNA moleculecomprising a DNA sequence which encodes a mammalian alpha kinaseselected from the group consisting of: a. the DNA sequence of SEQ ID NO:34; b. DNA sequences that encoding the amino acid sequence encoded bythe DNA sequence of subpart(a)and c. a fragment of SEQ ID NO: 34 whereinsaid fragment has alpha kinase activity; wherein said DNA sequence isoperatively linked to an expression control sequence.
 6. The unicellularhost of claim 5 wherein the unicellular host is selected from the groupconsisting of E. coli, Pseudomonas, Bacillus, Streptomyces, yeasts, CHO,R1.1, B-W, L-M, COS 1, COS 7, BSC1, BSC40, and BMT10 cells, plant cells,insect cells, mouse cells and human cells in tissue culture.
 7. Anisolated nucleic acid encoding a mammalian alpha kinase comprising theamino acid sequence set out in SEQ ID NO: 35, expressed in the heart andhaving alpha kinase activity.